Universal donor stem cells and related methods

ABSTRACT

Disclosed herein are universal donor stem cells and related methods of their use and production. The universal donor stem cells disclosed herein are useful for overcoming the immune rejection in cell-based transplantation therapies. In certain embodiments, the universal donor stem cells disclosed herein do not express one or more MHC-I and MHC-II human leukocyte antigens. Similarly, in certain embodiments, the universal donor stem cells disclosed herein do not express one or more human leukocyte antigens (e.g., HLA-A, HLA-B and/or HLA-C) corresponding to MHC-I and MHC-II human leukocyte antigens, thereby rendering such cells hypoimmunogenic.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/572,776, filed Nov. 8, 2017, which is a national stage filingunder 35 U.S.C. 371 of International Application No. PCT/US2016/031551,filed May 9, 2016, which claims the benefit of U.S. ProvisionalApplication No. 62/158,999, filed on May 8, 2015, the contents of whichare hereby incorporated by reference in its entirety. InternationalApplication No. PCT/US2016/031551 was published under PCT Article 21(2)in English.

BACKGROUND OF THE INVENTION

Degenerative diseases pose a disproportionate threat to human health.Often age-related, these diseases result in the progressivedeterioration of affected tissues and organs and, ultimately, disabilityand death of the affected subject. The promise of regenerative medicineis to replace diseased or missing cells with new healthy cells. Over thepast five years, a new paradigm for regenerative medicine hasemerged—the use of human pluripotent stem cells (hPSCs) to generate anyadult cell type for transplantation into patients. In principle,hPSC-based cell therapies have the potential to treat most if not alldegenerative illnesses, however the success of such therapies may belimited by a subject's immune response.

Strategies that have been considered to overcome the immune rejectioninclude HLA-matching (e.g. identical twin or umbilical cord banking),the administration of immunosuppressive drugs to the subject, blockingantibodies, bone marrow suppression/mixed chimerism, HLA-matched stemcell repositories and autologous stem cell therapy. Needed are novelapproaches, compositions and methods for overcoming immune rejectionassociated with cell replacement therapies.

SUMMARY OF THE INVENTION

Disclosed herein are efficient strategies to overcome immune rejectionin cell-based transplantation therapies by the creation of universaldonor stem cell lines that, as quality-controlled cell-based products,will form the base for regenerative cell therapies.

The present inventors have successfully employed genome editing toolssuch as a TALEN and/or CRISPR system in human pluripotent stem cells toreduce expression or knock out the highly polymorphic classical MHC-Igenes (HLA-A, HLA-B and HLA-C) and/or MHC-II genes. In certain aspects,such reduced expression or knock out of the MHC-I and/or MHC-II genes isaccomplished by directly and/or indirectly targeting the NLRC5, B2M andCIITA genes and other components of the MHC enhanceosome (e.g.,transcriptional regulators of MHC-I or MHC-II).

Disclosed herein are methods of preparing hypoimmunogenic stem cells,the method comprising modulating expression of one or more MHC-I andMHC-II human leukocyte antigens by the stem cell and thereby preparingthe hypoimmunogenic stem cell. Also disclosed are methods of modulatingexpression of one or more MHC-I and MHC-II human leukocyte antigens by astem cell, comprising deleting one or more genes encoding one or moretranscriptional regulators of MHC-I or MHC-II from at least one alleleof the cell and thereby modulating expression of the one or more MHC-Iand MHC-II human leukocyte antigens.

In certain aspects, modulating expression of the one or more MHC-I andMHC-II human leukocyte antigens comprises reducing, inhibiting and/orinterfering with the expression of the one or more MHC-I and MHC-IIhuman leukocyte antigens. In certain embodiments, modulating expressionof the one or more MHC-I and MHC-II human leukocyte antigens comprisesdeleting one or more genes encoding one or more transcriptionalregulators of MHC-I or MHC-II from at least one allele of the cell. Forexample, in certain embodiments such methods comprise deleting one ormore genes encoding one or more of the transcriptional regulators ofMHC-I or MHC-II selected from the group consisting of NLRC5, CIITA, B2Mand combinations thereof.

In certain aspects, the methods disclosed herein further comprisemodulating expression of one or more tolerogenic factors by the stemcell. Modulating expression of the tolerogenic factors may comprisesincreasing the expression of the tolerogenic factors. Such tolerogenicfactors may be inserted into a safe harbor locus (e.g., the AAVS1 locus)of at least one allele of the cell. In certain embodiments, suchtolerogenic factors inhibit immune rejection. In certain embodiments,such tolerogenic factors are selected from the group consisting ofHLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, CD47, C1-inhibitor, and IL-35.

Also disclosed herein are methods of preparing a hypoimmunogenic stemcell, the method comprising modulating expression of one or moretolerogenic factors by the stem cell and thereby preparing thehypoimmunogenic stem cell. In certain embodiments, modulating expressionof the tolerogenic factors comprises increasing the expression of thetolerogenic factors. Such tolerogenic factors may be inserted into asafe harbor locus (e.g., the AAVS1 locus) of at least one allele of thecell. In certain embodiments, such tolerogenic factors are selected fromthe group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, CD47,C1-inhibitor, and IL-35. In certain embodiments, such tolerogenicfactors inhibit immune rejection of the stem cell or of a differentiatedstem cell prepared therefrom.

In certain aspects, the methods disclosed further comprise modulatingexpression of one or more MHC-I and MHC-II human leukocyte antigens bythe cell, for example, by deleting one or more genes encoding one ormore transcriptional regulators of MHC-I or MHC-II from at least oneallele of the cell. In certain embodiments, transcriptional regulatorsof MHC-I or MHC-II from are selected from the group consisting of NLRC5,CIITA, B2M and combinations thereof.

Also disclosed herein are human stem cells that do not express NLRC5(e.g., a NLRC5^(−/−) knockout mutant stem cell). Similarly, alsodisclosed are human stem cells that do not express CIITA (e.g., a CIITAknockout mutant stem cell). Also disclosed are human stem cells that donot express B2M (e.g., a B2M^(−/−) knockout mutant stem cell). Incertain embodiments, also provided are human stem cells that do notexpress one or more of NLRC5, CIITA and B2M. In still other embodiments,also disclosed are human stem cells that do not express one or more ofHLA-A, HLA-B and HLA-C.

In certain embodiments, the stem cells disclosed herein expresses one ormore tolerogenic factors (e.g., one or more tolerogenic factors thatinhibit immune rejection). In certain aspects, such tolerogenic factorsare inserted into a safe harbor locus (e.g., the AAVS1 locus) of atleast one allele of the cell. In certain embodiments, the tolerogenicfactors are selected from the group consisting of HLA-C, HLA-E, HLA-G,PD-L1, CTLA-4-Ig, CD47, C1-inhibitor, and IL-35.

In some embodiments, the cell is an embryonic stem cell. In certainembodiments, the cell is a pluripotent stem cell. In certainembodiments, the stem cell is hypoimmunogenic.

In some embodiments, the cell has reduced MHC-I expression relative tothe original genotype or relative to a wild-type human stem cell. Incertain embodiments, the cell has reduced MHC-II expression relative tothe original genotype or relative to a wild-type human stem cell. Forexample, such cells may have reduced expression of one or more of HLA-A,HLA-B and HLA-C relative to the original genotype or relative to awild-type human stem cell.

Also disclosed herein are human stem cells that have been altered suchthat they comprise one or more tolerogenic factors inserted into a safeharbor locus (e.g., the AAVS1 locus) of at least one allele of the cell.In certain embodiments, such tolerogenic factors inhibit immunerejection. In certain embodiments, the tolerogenic factors are selectedfrom the group consisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig,CD47, C1-inhibitor, and IL-35. In certain aspects, such cells do notexpress one or more of NLRC5, CIITA and B2M. In certain aspects, suchcells do not express one or more of HLA-A, HLA-B and HLA-C. In certainembodiments, such cells have reduced expression of one or more of HLA-A,HLA-B and HLA-C relative to a wild-type human stem cell.

Also disclosed herein are methods of using the hypoimmunogenic cellsdisclosed herein for cell replacement therapy. For example, theuniversal stem cells disclosed herein may be incubated under appropriateconditions and differentiated into hypoimmunogenic cardiomyocytes,endothelial cells, hepatocytes or pancreatic beta cells.

Also disclosed herein are methods for producing hypoimmunogenic stemcells, the method comprising contacting a stem cell with a Cas proteinor a nucleic acid sequence encoding the Cas protein and a first pair ofribonucleic acids having sequences selected from the group consisting ofSEQ ID NOs: 36353-81239, thereby editing the NLRC5 gene to reduce oreliminate NLRC5 surface expression and/or activity in the cell.

Also disclosed are methods for producing hypoimmunogenic stem cells, themethod comprising contacting a stem cell with a Cas protein or a nucleicacid sequence encoding the Cas protein and a first pair of ribonucleicacids having sequences selected from the group consisting of SEQ ID NOs:5184-36352, thereby editing the CIITA gene to reduce or eliminate CIITAsurface expression and/or activity in the cell.

Also disclosed are methods for producing hypoimmunogenic stem cells, themethod comprising contacting a stem cell with a Cas protein or a nucleicacid sequence encoding the Cas protein and a first pair of ribonucleicacids having sequences selected from the group consisting of SEQ ID NOs:81240-85644, thereby editing the B2M gene to reduce or eliminate B2Msurface expression and/or activity in the cell.

Also disclosed herein are methods for producing a hypoimmunogenic stemcell, the method comprising: (a) contacting a stem cell with a Casprotein or a nucleic acid sequence encoding the Cas protein and a firstpair of ribonucleic acids having sequences selected from the groupconsisting of SEQ ID NOs: 36353-81239, thereby editing the NLRC5 gene toreduce or eliminate NLRC5 surface expression and/or activity in thecell; (b) contacting a stem cell with a Cas protein or a nucleic acidsequence encoding the Cas protein and a second pair of ribonucleic acidshaving sequences selected from the group consisting of SEQ ID NOs:5184-36352, thereby editing the CIITA gene to reduce or eliminate CIITAsurface expression and/or activity in the cell; and/or (c) contacting astem cell with a Cas protein or a nucleic acid sequence encoding the Casprotein and a third pair of ribonucleic acids having sequences selectedfrom the group consisting of SEQ ID NOs: 81240-85644, thereby editingthe B2M gene to reduce or eliminate B2M surface expression and/oractivity in the cell.

Also disclosed are methods for producing hypoimmunogenic stem cells, themethod comprising contacting a stem cell with a Cas protein or a nucleicacid sequence encoding the Cas protein and a first ribonucleic acidhaving a sequence selected from the group consisting of SEQ ID NOs:36353-81239, thereby editing the NLRC5 gene to reduce or eliminate NLRC5surface expression and/or activity in the cell.

Also disclosed are methods for producing hypoimmunogenic stem cells, themethod comprising contacting a stem cell with a Cas protein or a nucleicacid sequence encoding the Cas protein and a first ribonucleic acidhaving a sequence selected from the group consisting of SEQ ID NOs:5184-36352, thereby editing the CIITA gene to reduce or eliminate CIITAsurface expression and/or activity in the cell.

Also disclosed are methods for producing hypoimmunogenic stem cells, themethod comprising contacting a stem cell with a Cas protein or a nucleicacid sequence encoding the Cas protein and a first ribonucleic acidhaving a sequence selected from the group consisting of SEQ ID NOs:81240-85644, thereby editing the B2M gene to reduce or eliminate B2Msurface expression and/or activity in the cell.

Also disclosed herein are methods for producing hypoimmunogenic stemcells, the method comprising: (a) contacting a stem cell with a Casprotein or a nucleic acid sequence encoding the Cas protein and a firstribonucleic acid having a sequence selected from the group consisting ofSEQ ID NOs: 36353-81239, thereby editing the NLRC5 gene to reduce oreliminate NLRC5 surface expression and/or activity in the cell; (b)contacting a stem cell with a Cas protein or a nucleic acid sequenceencoding the Cas protein and a second ribonucleic acid having a sequenceselected from the group consisting of SEQ ID NOs: 5184-36352, therebyediting the CIITA gene to reduce or eliminate CIITA surface expressionand/or activity in the cell; and/or (c) contacting a stem cell with aCas protein or a nucleic acid sequence encoding the Cas protein and athird ribonucleic acid having a sequence selected from the groupconsisting of SEQ ID NOs: 81240-85644, thereby editing the B2M gene toreduce or eliminate B2M surface expression and/or activity in the cell.

Also disclosed herein are hypoimmunogenic stem cells comprising amodified genome comprising a first genomic modification in which theNLRC5 gene has been edited to reduce or eliminate NLRC5 surfaceexpression and/or activity in the cell by contacting the cell with a Casprotein or a nucleic acid encoding a Cas protein and a ribonucleic acidhaving a sequence selected from the group consisting of SEQ ID NOs:36353-81239.

Also disclosed are hypoimmunogenic stem cells comprising a modifiedgenome comprising a first genomic modification in which the CIITA genehas been edited to reduce or eliminate CIITA surface expression and/oractivity in the cell by contacting the cell with a Cas protein or anucleic acid encoding a Cas protein and a ribonucleic acid having asequence selected from the group consisting of SEQ ID NOs: 5184-36352.

Also disclosed are hypoimmunogenic stem cells comprising a modifiedgenome comprising a first genomic modification in which the B2M gene hasbeen edited to reduce or eliminate B2M surface expression and/oractivity in the cell by contacting the cell with a Cas protein or anucleic acid encoding a Cas protein and a ribonucleic acid having asequence selected from the group consisting of SEQ ID NOs: 81240-85644.

Also disclosed herein are hypoimmunogenic stem cells comprising amodified genome comprising: (a) a first genomic modification in whichthe NLRC5 gene has been edited to reduce or eliminate NLRC5 surfaceexpression and/or activity in the cell by contacting the cell with a Casprotein or a nucleic acid encoding a Cas protein and a ribonucleic acidhaving a sequence selected from the group consisting of SEQ ID NOs:36353-81239; (b) a second genomic modification in which the CIITA genehas been edited to reduce or eliminate CIITA surface expression and/oractivity in the cell by contacting the cell with a Cas protein or anucleic acid encoding a Cas protein and a ribonucleic acid having asequence selected from the group consisting of SEQ ID NOs: 5184-36352;and/or (c) a third genomic modification in which the B2M gene has beenedited to reduce or eliminate B2M surface expression and/or activity inthe cell by contacting the cell with a Cas protein or a nucleic acidencoding a Cas protein and a ribonucleic acid having a sequence selectedfrom the group consisting of SEQ ID NOs: 81240-85644.

Also disclosed are hypoimmunogenic stem cells comprising a modifiedgenome comprising a first genomic modification in which the NLRC5 genehas been edited to delete a first contiguous stretch of genomic DNA,thereby reducing or eliminating NLRC5 surface expression and/or activityin the cell, wherein the first contiguous stretch of genomic DNA hasbeen deleted by contacting the cell with a Cas protein or a nucleic acidencoding a Cas protein and a first pair of ribonucleic acids havingsequences selected from the group consisting of SEQ ID NOs: 36353-81239.

Also disclosed are hypoimmunogenic stem cells comprising a modifiedgenome comprising a first genomic modification in which the CIITA genehas been edited to delete a first contiguous stretch of genomic DNA,thereby reducing or eliminating CIITA surface expression and/or activityin the cell, wherein the first contiguous stretch of genomic DNA hasbeen deleted by contacting the cell with a Cas protein or a nucleic acidencoding a Cas protein and a first pair of ribonucleic acids havingsequences selected from the group consisting of SEQ ID NOs: 5184-36352.

Also disclosed are hypoimmunogenic stem cells comprising a modifiedgenome comprising a first genomic modification in which the B2M gene hasbeen edited to delete a first contiguous stretch of genomic DNA, therebyreducing or eliminating B2M surface expression and/or activity in thecell, wherein the first contiguous stretch of genomic DNA has beendeleted by contacting the cell with a Cas protein or a nucleic acidencoding a Cas protein and a first pair of ribonucleic acids havingsequences selected from the group consisting of SEQ ID NOs: 81240-85644.

Also disclosed herein are hypoimmunogenic stem cells comprising amodified genome comprising: (a) a first genomic modification in whichthe NLRC5 gene has been edited to delete a first contiguous stretch ofgenomic DNA, thereby reducing or eliminating NLRC5 surface expressionand/or activity in the cell, wherein the first contiguous stretch ofgenomic DNA has been deleted by contacting the cell with a Cas proteinor a nucleic acid encoding a Cas protein and a first pair of ribonucleicacids having sequences selected from the group consisting of SEQ ID NOs:36353-81239; (b) a second genomic modification in which the CIITA genehas been edited to delete a first contiguous stretch of genomic DNA,thereby reducing or eliminating CIITA surface expression and/or activityin the cell, wherein the first contiguous stretch of genomic DNA hasbeen deleted by contacting the cell with a Cas protein or a nucleic acidencoding a Cas protein and a first pair of ribonucleic acids havingsequences selected from the group consisting of SEQ ID NOs: 5184-36352;and/or (c) a third genomic modification in which the B2M gene has beenedited to delete a first contiguous stretch of genomic DNA, therebyreducing or eliminating B2M surface expression and/or activity in thecell, wherein the first contiguous stretch of genomic DNA has beendeleted by contacting the cell with a Cas protein or a nucleic acidencoding a Cas protein and a first pair of ribonucleic acids havingsequences selected from the group consisting of SEQ ID NOs: 81240-85644.

The above discussed, and many other features and attendant advantages ofthe present inventions will become better understood by reference to thefollowing detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 illustrates the promise of regenerative medicine.

FIGS. 2A-2B graphically depict the HLA barrier in transplantation. FIG.2A graphically illustrates MHC-I and MHC-II surface molecules thatdefine donor compatibility during organ transplant. FIG. 2B illustratesthe HLA region 6p21.1-21.3.

FIGS. 3A-3B illustrate targeting of transcriptional regulators ofantigen presentation. FIG. 3A illustrates a model of the MHC-IIenhanceosome. FIG. 3B depicts a model of the NLRC5 enhanceosome at theMHC-I promoter.

FIG. 4 illustrates MHC-I expression in WT HuES9 and the indicatedmodified mutant cell lines. Cells were stimulated for 48 hrs. with IFNγand MHC-I expression was assessed by FACS.

FIGS. 5A-5C illustrate that CIITA-deficient stem cell-derived macrophagelack MHC-II expression. FIG. 5A depicts a time line of macrophagedifferentiation.

FIG. 5B shows a bright-field image of stem cell-derived macrophages onday 5 of M-CSF medium. FIG. 5C shows MHC-II expression in inducedmacrophages, as assessed by qPCR 48 hrs. post stimulation with IFNγ, andevidences reduced MHC-II expression relative to wild-type cells. Asillustrated in FIG. 5C, MHC-II expression can be efficiently abrogatedby targeting the first coding exon of the CIITA gene depicted in FIGS.10A-10B.

FIG. 6 illustrates a time line of the teratoma rejection study disclosedherein in which humanized NSG mice—BLT mice are given 3 months to fullyreconstitute a human immune system before injection of the indicatedgenome-edited stem cell lines.

FIG. 7 demonstrates improved engraftment of genome-edited stem cells inhumanized mice. Illustrated are the quantification of teratoma typeobserved following the teratoma rejection study depicted in FIG. 6 .Upon review of the morphology of the teratoma samples, a clear phenotypethat correlates with the levels of MHC-I expression was observed.

FIGS. 8A-8C demonstrate that proliferation of infiltrating CD8+ T cellindicates immune rejection. FIGS. 8A and 8B illustrate immunostaining ofteratoma-infiltrating CD3+ lymphocytes. FIG. 8C illustrates co-stainingof CD8 and Ki67, which is a proliferation marker, and highlightsproliferating CD8+ T cells (white arrows). Nuclear DAPI staining isshown in blue.

FIGS. 9A-9D demonstrate that the deletion of NLRC5 using TALENs resultsin reduced MHC-I expression in induced pluripotent stem cell-derivedhepatocyte-like cells (HLCs). FIG. 9A illustrates the NLRC5 locus. FIG.9B shows targeting of the NLRC5 locus to abrogate NLRC5 expression.FIGS. 9C-9D illustrate reduced MHC-I expression in NLTC5^(−/−) HLCs.

FIGS. 10A-10B illustrate targeting of the first coding exon of the CIITAgene. FIG. 10A depicts the CIITA locus. FIG. 10B shows the targeting ofthe CIITA locus to abrogate MHC-II expression.

FIGS. 11A-11C demonstrate that tolerogenic factors, such as PD-L1 andHLA-G, can be expressed from a safe harbor locus.

FIG. 12 shows an exemplary amino acid sequence of a Cas protein. Yellowhighlights indicate Ruv-C-like domain. Underlining indicates HNHnuclease domain.

FIG. 13 illustrates a comparison of the MHC-II and MHC-I enhanceosomes.

FIGS. 14A-14J demonstrates the targeting of HLA-A, HLA-B and HLA-C.

FIG. 14A depicts an HLA-B and HLA-C knock-out strategy. Two short guideRNAs (sgRNAs) were designed upstream of the HLA-B locus and downstreamof HLA-C, which allow excision of the HLA-B and HLA-C genes. sgRNA #1and sgRNA #2 target the HLA-B upstream region, and sgRNA #3 and sgRNA #4target HLA-C the downstream region. FIG. 14B shows that PCR screeningconfirms clone 1D as a homozygous knock-out clone. FIG. 14B includes aschematic showing the PCR verification strategy for successful deletionof HLA-B and HLA-C. Two pairs of wild-type (WT) primers were designedflanking each cutting site, with predicted amplicon sizes of 545 bp and472 bp. Clone 1D was identified as a homozygous knock-out clone by thepresence of about 680 bp PCR band generated with KO primers and theabsence of bands using the two different sets of WT primers. Genomic DNAwas isolated from the indicated targeted HuES8 clones. FIG. 14C providesa schematic showing sequence confirmation of HLA-B, HLA-C deletion in 1DKO clone. The sequencing results of 1D PCR product demonstratedsuccessful deletion of HLA-B and HLA-C genes in HuES8. FIG. 14D showsthat RT-PCR confirms the loss of HLA-B and HLA-C mRNA expression inHuES8 cells. RT-PCR with HLA-B and HLA-C specific primers demonstratedthe mRNA expressions of HLA-B and HLA-C in clone 1D are eliminated.GAPDH, TOP1 and HPRT1 were used as internal controls. FIG. 14E providesa schematic showing sequence confirmation of HLA-B RT-PCR bands obtainedfrom WT and clone 1D. WT and 1D RT-PCR products amplified with HLA-Bprimers were sequenced and identified as HLA-B and HLA-A mRNAs,respectively using BLAST. These results demonstrated that in the absenceof the HLA-B gene, the HLA-B specific primers will amplify HLA-A mRNA inHuES8 clone 1D. FIG. 14F shows the karyotyping of HuES8 clone 1D byNanoString nCounter. HuES8 clone 1D displays a normal karyotype asassessed by NanoString nCounter set. FIG. 14G provides a schematicshowing HLA-A knockout strategy using the dual sgRNA approach. Theschematic shows the positions of the two sgRNAs (sgRNA #5 and sgRNA #6)that were designed to bind upstream and downstream of HLA-A. FIG. 14Hshows the on-target cutting efficiency of HLA-A sgRNA using TIDE. Thecutting efficiency of sgRNA #5 and sgRNA #6 was determined in 293T cellsusing TIDE. FIG. 14I demonstrates that PCR screening confirmed clone 4Eas a heterozygous HLA-A knockout clone. PCR screening strategy confirmeddeletion of HLA-A in HuES8. KO primers were designed with one primerannealing upstream and one primer annealing downstream of the cuttingsites. Upon HLA-A deletion, the resulting amplicon is observed as 220 bpon a 2% agarose gel. Two pairs of WT primers were designed flanking eachcutting site, with predicted amplicons sizes of 589 bp and 571 bp. Clone4E was identified as heterozygous clone due to the presence of bandsgenerated with KO primers and WT primers amplified from genomic DNA.FIG. 14J includes a schematic showing that sequencing confirmssuccessful deletion of HLA-A in HuES8 cells. Sequencing of the PCRproduct amplified from genomic DNA of clone 4E using KO primersdemonstrates successful deletion of HLA-A in HuES8.

FIGS. 15A-15D show TALEN-induced CIITA and NLRC5 mutations in BJ-RIPSCsand HuES9 cells. FIG. 15A provides a schematic of TALEN-induced CIITAmutations in BJ-RIPSCs. FIG. 15B provides a schematic of TALEN-inducedNLRC5 mutations in BJ-RIPSCs. FIG. 15C provides a schematic ofTALEN-induced NLRC5 mutations in HuES9s. FIG. 15D provides a schematicof TALEN-induced CIITA mutations in HuES9s.

FIGS. 16A-16G demonstrate targeting of NLRC5 and CIITA utilizing aCRISPR system to achieve a reduction in MHC class I expression andcomplete loss of MHC class II expression. FIG. 16A shows reduced MHC-Iexpression in NLRC5−/− Thp1 cells, including a NLRC5 targeting strategyin Thp1 cells. The + indicates 48 hours of IFNγ stimulation. FIG. 16Bprovides schematics of multiple targeting strategies. FIG. 16B providesa schematic of targeting NLRC5 using CRISPR, a schematic of targetingB2M using CRISPR, a schematic of targeting CIITA using TALENs and aschematic of targeting CIITA using CRISPR. FIG. 16C shows reduced MHCclass I expression in HuES9 cells following targeting with NLRC5 or B2MCRISPRs. The graph shows low basal MHC-I expression in stem cells. TheMHC-I expression can be increased by IFNγ stimulation. About a 50%reduction of MHC-I expression occurs in IFNγ-treated NLRC5−/− cells. Acomplete loss of MHC-I surface expression is shown in B2M−/− cells. FIG.15D shows a lentiviral transduction of Thp-1. A dual-vector lentiviralGeCKO system is used. The two component system includesLenti-Cas9-Blasticidin, which acts to establish stable Thp-1 Cas9 cellline, and Lenti-Guide-puro, which acts to bring in the guide. FIG. 16Eidentifies various CRISPRs used for the lentiviral transduction ofThp-1. CRISPRs targeting CIITA, NLRC5 and IRF1 are provided. FIG. 16Fshows that CIITA and NLRC5 act independently on MHC-II and MHC-I,respectively. Thp1 was transduced with lentivirus encoding NLRC5 andCIITA. A B2M CRISPR is used as a positive control. All cells werestimulated ON with 50U IFNγ to boost HLA expression. HLA-A2 1:200; DR1:100. FIG. 16G shows targeting of IRF1 results in loss of MHC-IIexpression. Thp1, 10 days post CRISPR transduction. All cells werestimulated ON with 50U IFNγ. HLA-A2 1:200; DR 1:100.

FIGS. 17A-17K demonstrate targeting of IRF1 utilizing a CRISPR system toachieve reduced MHC class I expression in human pluripotent stem cells(HuES9) and Thp-1 cells. FIG. 17A shows a dual CRISPR strategy targetingthe IRF1 locus. Three different CRISPRs are identified. FIG. 17Bdemonstrates the testing of different IRF1 guide combinations. Screeningresults of different guide combinations are provided. FIG. 17Cdemonstrates a ‘dual guide strategy’ for the targeted deletion of IRF1.Screening results of the dual guide strategy are provided. FIG. 17Dprovides a schematic showing sequence confirmation of IRF-1 CRISPRinduced deletion. FIG. 17E provides the screening results of IRF-1targeted HuES9 cells. The screening is of a dual CRISPR strategy usingCRISPRs #2 and #3. The presence of the PCR band suggests successfultargeting. FIG. 17F demonstrated reconfirmation of IRF-1 clones.Screening results using primers KYM07 and TM377 are provided. FIG. 17Gdemonstrates genotypes of IRF-1 clones. Screening results are provided.FIG. 17H provides a schematic showing sequence confirmation of IRF-1CRISPR induced deletion in clone 12. FIG. 17I provides a schematicshowing sequence confirmation of IRF-1 CRISPR induced deletion in clone17. FIG. 17J provides a schematic showing sequence confirmation of IRF-1CRISPR induced deletion in clone 21. FIG. 17K shows impaired MHC class Iinduction in IRF1−/− HuES9 clones following IFNγ treatment. P42 are WT;C7 are B2M KO; + identifies 48 hours IFNγ treatment.

FIGS. 18A-18H demonstrate targeting of RFX5, RFX-ANK and RFX-AP in 293Tcells utilizing CRISPR results in reduced MHC class I expression. FIG.18A demonstrates dual guide targeting of RFX5 in 293T cells.Combinations of four different CRISPRs were examined. FIG. 18B showsthat the targeting of RFX5 results in reduced MHC class I expression.FIG. 18C demonstrates dual guide targeting of RFX-ANK in 293T cells.Combinations of four different CRISPRs were examined FIG. 18D shows thatthe targeting of RFX-ANK results in reduced MHC class I expression. FIG.18E shows that targeting RFX5 and RFX-ANK results in reduced MHC class Iexpression. FIG. 18F provides a bar graph showing that targeting RFX5and RFX-ANK results in reduced MHC class I expression. FIG. 18Gdemonstrates dual guide targeting of RFX-AP in 293T cells. Combinationsof four different CRISPRs were examined. FIG. 18H shows that thetargeting of RFX-AP results in reduced MHC class I expression.

FIGS. 19A-19E demonstrate targeting of NFY-A, NFY-B and NFY-C utilizingCRISPR results in reduced MHC class I expression. FIG. 19A demonstratesdual guide targeting of NFY-A in 293T cells. Combinations of fourdifferent CRISPRs were examined FIG. 19B demonstrates dual guidetargeting of NFY-C in 293T cells. Combinations of four different CRISPRswere examined. FIG. 19C shows that the targeting of NFY-C and NFY-Aresults in reduced MHC class I expression. FIG. 19D demonstrates dualguide targeting of NFY-B in 293T cells. Combinations of four differentCRISPRS were examined FIG. 19E shows that the targeting of NFY-B resultsin reduced MHC class I expression.

FIGS. 20A-20D show that surface trafficking of MHC class I molecules canbe suppressed by disrupting the TAP1 gene, an ER-resident peptidetransporter. FIG. 20A shows TAP1 CRISPR expression reduces MHC-I surfaceexpression in Jurkat T cells. Combinations of four different CRISPRs wasexamined FIG. 20B demonstrates TAP1 CRISPR expression reduces MHC-Isurface expression in Jurkat T cells. FIG. 20C demonstrates eliminatingHLA surface expression in Jurkat (Cas9) T cells. The Jurkat cell linewas established from the peripheral blood of a 14 year old having acuteT cell leukemia by Schneider et al. FIG. 20D shows eliminating HLAsurface expression in Jurkat (Cas9) T cells. The cells were treated withIFNγ for 48 hours.

FIGS. 21A-21D demonstrate the use of a CRISPR gRNA identified as an HLARazor that allows simultaneous deletion of all MHC class I alleles bytargeting a conserved region in the HLA genes. FIG. 21A provides aschematic showing the targeting of a conserved sequence found in allHLAs by CRISPR or TALENs using an HLA Razor. The two violet boxesindicate the binding sites for the panHLA TALEN pair. The blue arrowindicates the pan-HLA CRISPR tested; the PAM is boxed in blue. FIG. 21Bshows the expression of pan-HLA TALENs in 293T and HuES9 cells 72 hourspost transfection. FIG. 21C shows HLA-Razor CRISPR blunts MHC class Iexpression. The 293T cells were co-transfected with Cas9-GFP. Theresults are shown 72 hours post transfection. FIG. 21D provides aschematic showing a comparison of the activity of two different HLARazors. Two HLA Razor guides are identified.

FIGS. 22A-22F show the results of a PD-L1 and HLA-G knock-in strategy.FIG. 22A provides a schematic showing PD-L1 and HLA-G knock-in strategy.WT primers and knock-in (KI) primers for clone screening were designed.The amplicon with WT primers is predicted as 488 bp, and the ampliconswith KI primers are predicted as 403 bp and 915 bp. FIG. 22B depicts thedesign of the knock-in donor plasmid. The design of the knock-in donorplasmid shows that the reading frames of PD-L1 and HLA-G are linked byT2A and their expression is driven by a CAGGS promoter. Puromycin wasused as a drug resistance marker following the SA-2A gene trap element.FIG. 22C demonstrates ectopic PD-L1 and HLA-G expression in 293T cells.The expression of PD-L1 and HLA-G were examined in the donorplasmid-transfected 293T cells by FACS analysis. APC-conjugated PD-L1antibody and FITC-conjugated HLA-G antibody were used. FIG. 22D showsectopic HLA-G expression in JEG-3 cells. Donor plasmid was transfectedinto an HLA-G−/−JEG-3 cell line, and ectopic HLA-G expression wasexamined by FACS analysis 48 hours post-transfection. A PE-conjugatedHLA-G antibody (MEM/G9) was used to detect surface HLA-G surfaceexpression. FIG. 22E provides PCR screening results that confirm clone1G as a heterozygous KI clone for PD-L1/HLA-G. Clone 1G was identifiedas a heterozygous KI clone by the presence of bands using both WTprimers and KI-specific primers amplified from genomic DNA of targetedHuES8 cells. FIG. 22F shows stable PD-L1 and HLA-G expression from asafe harbor locus in HuES8. The expression of PD-L1 was verified inHuES8 KI clone 1G by FACS analysis using an APC-conjugated PD-L1antibody.

FIG. 23 demonstrates expression of CD47 in 293T cells and hPSCs using aknock-in strategy. Human CD47 was cloned into an expression plasmiddriven by a CAG promoter, which in addition contains an IRES-GFP. 293Tcells already express high levels of CD47, the human ‘don't eat me’signal, which prevents engulfment of cells from macrophages. Expressioncan be increased by overexpression of CD47 in both 293T cells, as wellas in human pluripotent stem cells (HuES9), as detected by FACS using aCD47-specific antibody. Overexpression of CD47 will assist engraftmentof stem cell-derived transplants by protecting cells from macrophageengulfment.

FIG. 24 demonstrates that deletion of TRAC and TRBC in HuES9 disruptsTCR expression. A dual guide RNA approach was used to introducedeletions into the TRAC and TRBC loci in HuES9 cells. TCRA wt band is249 bp, and is 209 bp after deletion. TCRB wt band is 162 bp, and is 140bp after deletion. Bands identified with a * are TCRB KO in HuES9 cells;bands identified with a ** are TCRA KO in HuES9 cells; and bandsidentified with a *** are TCRA KO in HuES9 B2M−/−CIITA−/− cells. TCRA KOin HuES9 B2M−/−CIITA−/− cells is a triple knock-out stem cell line forB2M−/−, CIITA−/− and TCR−/−. Upon differentiation into T cells thistriple knock out stem cell line will be devoid of MHC-I, MHC-II and TCRsurface expression.

FIGS. 25A-25D demonstrate targeting of CD274/B7-H1/PD-L1 in a variety ofcell lines. FIG. 25A identifies an example of a CRISPR guide sequencethat may be used when targeting CD274/B7-H1/PD-L1. CD274/B7-H1/PD-L1knock out cell lines are identified. FIG. 25B shows the screening oftargeted B7-H1 colonies in JEG-3 cells. The screening identified aCRISPR cutting efficiency of 22/265 or about 8.3%. FIG. 25C demonstratesthe reconfirmation of 501 melanoma knock-out clones. FIG. 25Ddemonstrates the reconfirmation of MalMe melanoma knock-out clones.

FIGS. 26A-26P demonstrate targeting of co-inhibitory/co-stimulatoryreceptors or their ligands. For each target, the indicated four CRISPRshave been cloned and tested for on-target activity in 293T cells. Theexpected size of the PCR band, when cutting of both CRISPRs occurs, isindicated below the gel picture. FIG. 26A showsco-stimulatory/co-inhibitory molecules and their receptors on T cells.

FIG. 26B demonstrates dual guide targeting of TIGIT in 293T cells. Allfour CRISPRS were found to work. FIG. 26C demonstrates dual guidetargeting of TIM3 in 293T cells. All four CRISPRs were found to work.FIG. 26D demonstrates dual guide targeting of HVEM in 293T cells. Allfour CRISPRs were found to work. FIG. 26E demonstrates dual guidetargeting of 2B4/CD244 in 293T cells. All four CRISPRs were found towork. FIG. 26F demonstrates dual guide targeting of CD28 in 293T cells.CRISPRs #1, #2 and #3 were found to work. FIG. 26G demonstrates dualguide targeting of OX40 in 293T cells. CRISPRs #1, #2 and #4 were foundto work. FIG. 26H demonstrates dual guide targeting of B71 in 293Tcells. CRISPRs #1, #3 and #4 were found to work. FIG. 26I demonstratesdual guide targeting of CD226 in 293T cells. CRISPRs #1 and #2 werefound to work. FIG. 26J demonstrates dual guide targeting of CD2 in 293Tcells. CRISPRs #1, #2 and #3 were found to work. FIG. 26K demonstratesdual guide targeting of LAGS in 293T cells. CRISPRs #2 and #3 were foundto work. FIG. 26L demonstrates dual guide targeting of BTLA in 293Tcells. All four CRISPRs were found to work. FIG. 26M demonstrates dualguide targeting of ICOS in 293T cells. CRISPRs #2, #3 and #4 were foundto work. FIG. 26N demonstrates dual guide targeting of CD27 in 293Tcells. CRISPRs #1 and #2 were found to work. FIG. 26O demonstrates dualguide targeting of ST2 in 293T cells. All four CRISPRs were found towork. FIG. 26P demonstrates dual guide targeting of GITR in 293T cells.CRISPRs #1 and #3 were found to work.

FIG. 27 depicts various cell lines tested and with which targets thecells lines were tested. HuES8 and HuES9 are human ES cell lines.BJ-RiPSCs is an iPSC line. All other enhanceosome components not shownin the table (e.g., RFX and NFY) were only tested in HEK293T cells.

FIGS. 28A-28J provides that modified ES cells can be differentiated intovarious different cell types with reduced or absent HLA expression. FIG.28A provides that the modified ES call can be differentiated intoMesenchymal Progenitor Cells (MPCs), endothelial cells (ECs),macrophages, hepatocytes, beta-cells and neural progenitor cells (NPCs).FIG. 28B shows reduced MHC-I expression in NLRC5−/− human ES cells.There is low basal MHC-I expression in stem cells. The expression can beincreased by IFNγ stimulation. About a 50% reduction in MHC-I expressionoccurs in IFNγ-treated NLRC5−/− cells. FIG. 28C shows reduced MHC-Iexpression in NLRC5−/− human mesenchymal progenitor cells (MPCs). Acomparison is provided of HuES9 cells and MPCs. FIG. 28D shows reducedMHC-I expression in stem cell-derived NLRC5−/− endothelial cells (ECs).FIG. 28E demonstrates that ECs exhibit similar differentiationefficiency. FIG. 28F shows loss of HLA expression in B2M−/−CIITA−/−ECs.N=3, 48 hours of IFNγ treatment. FIG. 28G demonstrates reduced MHC classI expression in NLRC5−/− hepatocyte-like cells. A schematic is includeddemonstrating the targeting strategy and CRISPR design. The HLCs arederived from BJ-RiPSCs, day 7 of final hepatocyte differentiation. FIG.28H demonstrates that mutation of CIITA abrogates MHC class IIexpression in hESC-derived macrophages. A schematic is includeddemonstrating the targeting strategy and CRISPR design. TheHuES9-derived iMΦs, day 5 M-CSF.

FIG. 28I shows neural progenitor cell (NPC) differentiation.B2M−/−CIITA−/−HuES9 form Nestin+ neural rosettes (white arrow). FIG. 28Jdemonstrates the adaptation of stem cells to spin culture for beta-celldifferentiation. This is the first step in the beta-cell differentiationprotocol.

FIG. 29A-29B provide in vivo data in a teratoma model. FIG. 29A providesthat ‘hypoimmunogenic’ DKO cell lines were generated. The cell linesgenerated include WT HuES9, NLRC5−/−CIITA−/− HuES9 and B2M−/−CIITA−/−HuES9. The WT cell lines exhibited MHC-I and MHC-II expression. TheNLRC5−/−CIITA−/− cells lines exhibited reduced MHC-I expression and noMHC-II expression. The B2M−/−CIITA−/− cell lines exhibited no MHC-I andMHC-II expression. FIG. 29B shows improved engraftment of genome-editedstem cells in humanized mice. The three cell lines tested were WT HuES9,NLRC5−/−CIITA−/− HuES9 and B2M−/−CIITA−/− HuES9.

FIGS. 30A-30D demonstrates CRISPR targeting of B7-H3 in JEG-3 cells.FIG. 30A provides a CRISPR guide sequence for targeting B7-H3. FIG. 30Bshows a screening of targeted B7-H3 colonies in JEG-3 cells. The CRISPRcutting efficiency is shown to be 15/80 or about 18.7%. FIG. 30C showsconfirmation of B7-H3 knock-outs through sequencing. FIG. 30Ddemonstrates loss of B7-H3 surface expression in targeted JEG3 clones.

FIG. 31 shows loss of MHC class I surface expression in B2M−/− JurkatCas9 T cells. The B2M−/− Jurkat Cas9 T cells are compared to Jurkat Cas9T cells that do not include a knock out of B2M.

FIGS. 32A-32H demonstrate targeting clinically relevant loci in humancells using CRISPR/Cas9. FIG. 32A is a schematic of gRNAs targeting B2M.FIG. 32B is a histogram of B2M surface expression in HEK293T cells. FIG.32C shows B2M deletion efficiency with various gRNAs in HEK293T cells;n=3 (mean±SEM). FIG. 32D is a schematic of gRNAs targeting CCR5. Orangeand green arrows represent primer pairs used to amplify the region foranalysis. FIG. 32E shows results of Surveyor assays of each gRNAtargeting CCR5 in K562 cells. % InDels is indicated under each guide.FIG. 32F illustrates B2M deletion efficiency of selected gRNAs inprimary CD4+ T cells in comparison to 293T cells; n=6 (mean±SEM). FIG.32G shows results of surveyor assay of crCCR5_A and crCCR5_B targetingCCR5 in K562 cells and HSPCs. FIG. 32H illustrates clonal deletionefficiency of crCCR5_A and crCCR5_B targeting of CCR5 in HSPCs (n=2) asdetermined by Sanger sequencing. (Note: crB2M_14 is not depicted inpanel A schematic, as it is located 20 Kb downstream of codingsequence.).

FIGS. 33A-33E demonstrate an evaluation of on target mutationalefficiencies of various gRNAs targeting B2M. FIG. 33A shows B2M deletionefficiency for all gRNAs targeting B2M locus in HEK293T cells asmeasured by flow cytometry. Pooled data from 3 independent experimentsshown as mean±SEM. FIG. 33B shows B2M deletion efficiencies of selectedguides in HEK293T cells, measured as % InDels by CEL Surveyor assay.FIG. 33C is a comparison of B2M surface expression in HEK293T cells andprimary CD4+ T cells when transfected with Cas9 and guide crB2M_13. FIG.33D shows B2M deletion efficiency for selected guides targeting the B2Mlocus in primary CD4+ T-cells, as measured by flow cytometry. FIG. 33Eshows B2M deletion efficiencies of selected guides in primary CD4+ Tcells, measured as % InDels by CEL Surveyor assay.

FIGS. 34A-34E depict a dual gRNA approach for CRISPR/Cas9 genome editingin primary human hematopoietic stem and effector cells. FIG. 34A is aschematic of dual gRNA approach for targeting the B2M locus. gRNA pairsare in red. The offset in base pairs between Cas9 sites for each gRNAcombination (right panel). FIG. 34B shows B2M deletion efficiency inCD4+ T cells for 6 dual gRNA combinations (n=3; mean±SEM). FIG. 34C is aFACS plots showing loss of B2M expression of either crB2M_13 or crB2M_8alone or in combination in primary CD4+ T cells. FIG. 34D is a schematicof dual gRNA approach for targeting CCR5. gRNA pairs are shown in red.Orange and green arrowheads represent primer pairs used to amplify theregion. The offset between the Cas9 sites of each gRNA pair (rightpanel). FIG. 34E is a gel electrophoresis image of CD34+ HSPCs derivedclones targeted with crCCR5_D+Q analyzed by PCR. Note the deletion ofthe 205 bp region between the two gRNA cutting sites (top panel; WT:wild type; ΔCCR5: deleted; green * denotes a WT clone; orange * denotesa heterozygous clone; and red * denotes a homozygous deleted clone).Clonal deletion efficiency for three dual gRNA combinations targetingCCR5 in CD34+ HSPCs (n=4; % mean±SEM; bottom panel).

FIGS. 35A-35G demonstrate the targeting efficiency of dual gRNAcombinations. FIG. 35A shows B2M deletion efficiency for 6 dual gRNAcombinations from three independent donors as measured by flowcytometry. FIG. 35B are FACS plots showing loss of MHC class I surfaceexpression (bottom panel) following B2M deletion (top panel). FIG. 35Cis a schematic of the single cell nested PCR strategy for the B2M locus(left panel), black and gray arrowheads: control primer pairs, orangeand green arrowheads: primer pairs flanking targeting region. % B2M nullsingle cells is shown (right panel, n=301). FIG. 35D is a Sangersequencing chromatogram showing predicted deletion of targeted region atB2M locus. FIG. 35E shows clonal CCR5 deletion efficiency for three dualgRNA combinations in CD34+ HSPC-mPB obtained from multiple donors. DNAisolated from individual colony was analyzed by PCR and gelelectrophoresis. FIG. 35F is a schematic of the single cell nested PCRstrategy (left panel) for determining deletion of CCR5 in primary CD4+ Tcells. % CCR5 null single cells is shown (right panel, n=363). FIG. 35Gshows Sanger sequencing chromatogram shows predicted deletion attargeted region.

FIG. 36 demonstrates B2M deletion efficiencies of selected guides in293T cells. Arrows on the Surveyor assays show nuclease cleavage bonds.

FIG. 37 demonstrates a comparison of B2M surface expression in 293Tcells when transfected with AsCpf1 and guide crB2M.

FIG. 38 demonstrates a comparison of B2M surface expression in 293Tcells when transfected with LbCpf1 and guide crB2M.

FIG. 39 depicts Cpf1 crRNA design and cloning information.

FIGS. 40A-40G demonstrate generation and characterization of B2M KO JEG3cells using TALENs. FIG. 40A depicts a design of B2M TALEN and inducedmutations. FIG. 40B depicts an analysis of B2M at the transcript andprotein levels. FIG. 40C demonstrates an analysis of B2M at the surfaceexpression level. FIG. 40D demonstrates that AB2M clones are devoid ofMHC-I surface expression. FIG. 40E demonstrates that AB2M clones aredevoid of HLA-G surface expression. FIG. 40F demonstrates that AB2Mclones are devoid of HLA-C surface expression. FIG. 40G demonstratesthat AB2M clones are devoid of HLA-E surface expression.

FIG. 41 demonstrates an exemplary TCR targeting strategy of the presentinvention. FIG. 41 is a schematic illustration depicting the location ofthe CRISPR gRNAs targeting the first coding exons of the TCRalpha andTCRbeta chains, respectively.

FIGS. 42A-42B demonstrate deletion of T cell receptor in Jurkat T cells.FIG. 42A depicts the results of a FACS analysis employing an anti-CD3antibody, which reveals successful TCR deletion in Jurkat T cells thatstably express the Cas9 nuclease. FIG. 42B shows the results of aSURVEYOR™ assay confirming cutting at the TCRa and TCRb loci.

FIGS. 43A-43B demonstrate TCR Deletion in Primary Human CD3+ T Cells.FIG. 43A shows the results of a SURVEYOR™ assay demonstrating CRISPRcutting at the TCRa and TCRb loci in CD3+ T cells obtained from twoindependent donors. FIG. 43B shows the loss of TCR surface expressiondemonstrated by FACS analysis.

FIGS. 44A-44C demonstrate an exemplary PD-1 locus targeting strategy ofthe present invention. FIG. 44A is a schematic representation of thePD-1 targeting strategy. FIG. 44B demonstrates that the double CRISPRstrategy results in cutting by both CRISPRs targeting the PD-1 locus inHEK293T cells. FIG. 44C is a schematic representation of sequencing,which confirmed the predicted deletion in the PD-1 locus aftertransfection of two CRISPRs targeting the PD-1 gene (PDCD1).

FIGS. 45A-35B demonstrate the loss of PD-1 expression in Jurkat T cells.FIG. 45A shows the results of FACS analysis, demonstrating the loss ofPD-1 expression in activated Jurkat T cells. FIG. 45B shows the resultsof a SURVEYOR™ assay confirming cutting at the PD-1 locus.

FIGS. 46A-46C demonstrate an exemplary CTLA4 locus targeting strategy ofthe present invention. FIG. 46A is a schematic representation of theCTLA4 targeting strategy. FIG. 46B demonstrates that the double CRISPRstrategy results in cutting by both CRISPRs targeting the CTLA4 locus inHEK293T cells. FIG. 46C is a schematic representation of sequencing,which confirmed the predicted deletion in the CTLA4 locus aftertransfection of two CRISPRs targeting the CTLA4 gene (CTLA4).

FIGS. 47A-47B demonstrate cutting at the CTLA-4 locus in Jurkat T cells.FIG. 47A demonstrates that the double CRISPR strategy results in cuttingby both CRISPRs targeting the CTLA4 locus in Jurkat T cells. FIG. 47Bshows the results of a SURVEYOR™ assay, demonstrating successful cuttingby both CTLA4 CRISPRs in Jurkat T cells.

The following Tables 8-54 are submitted herewith as Appendices 1-47,respectively:

Table 8—exemplary gRNA sequences useful for targeting HLA-A;

Table 9—exemplary gRNA sequences useful for targeting HLA-B;

Table 10—exemplary gRNA sequences useful for targeting HLA-C;

Table 11—exemplary gRNA sequences useful for targeting RFX-ANK;

Table 12—exemplary gRNA sequences useful for targeting CIITA;

Table 13—exemplary gRNA sequences useful for targeting NFY-A;

Table 14—exemplary gRNA sequences useful for targeting NLRC5;

Table 15—exemplary gRNA sequences useful for targeting B2M;

Table 16—exemplary gRNA sequences useful for targeting RFX5;

Table 17—exemplary gRNA sequences useful for targeting RFX-AP;

Table 18—exemplary gRNA sequences useful for targeting HLA-G;

Table 19—exemplary gRNA sequences useful for targeting HLA-E;

Table 20—exemplary gRNA sequences useful for targeting NFY-B;

Table 21—exemplary gRNA sequences useful for targeting PD-L1;

Table 22—exemplary gRNA sequences useful for targeting NFY-C;

Table 23—exemplary gRNA sequences useful for targeting IRF1;

Table 24—exemplary gRNA sequences useful for targeting TAP1;

Table 25—exemplary gRNA sequences useful for targeting GITR;

Table 26—exemplary gRNA sequences useful for targeting 41BB;

Table 27—exemplary gRNA sequences useful for targeting CD28;

Table 28—exemplary gRNA sequences useful for targeting B7-1;

Table 29—exemplary gRNA sequences useful for targeting CD47;

Table 30—exemplary gRNA sequences useful for targeting B7-2;

Table 31—exemplary gRNA sequences useful for targeting OX40;

Table 32—exemplary gRNA sequences useful for targeting CD27;

Table 33—exemplary gRNA sequences useful for targeting HVEM;

Table 34—exemplary gRNA sequences useful for targeting SLAM;

Table 35—exemplary gRNA sequences useful for targeting CD226;

Table 36—exemplary gRNA sequences useful for targeting ICOS;

Table 37—exemplary gRNA sequences useful for targeting LAGS;

Table 38—exemplary gRNA sequences useful for targeting TIGIT;

Table 39—exemplary gRNA sequences useful for targeting TIM3;

Table 40—exemplary gRNA sequences useful for targeting CD160;

Table 41—exemplary gRNA sequences useful for targeting BTLA;

Table 42—exemplary gRNA sequences useful for targeting CD244;

Table 43—exemplary gRNA sequences useful for targeting LFA-1;

Table 44—exemplary gRNA sequences useful for targeting ST2;

Table 45—exemplary gRNA sequences useful for targeting HLA-F;

Table 46—exemplary gRNA sequences useful for targeting CD30;

Table 47—exemplary gRNA sequences useful for targeting B7-H3;

Table 48—exemplary gRNA sequences useful for targeting VISTA;

Table 49—exemplary gRNA sequences useful for targeting TLT;

Table 50—exemplary gRNA sequences useful for targeting PD-L2;

Table 51—exemplary gRNA sequences useful for targeting FOXP3;

Table 52—exemplary gRNA sequences useful for targeting CD58;

Table 53—exemplary gRNA sequences useful for targeting CD2; and

Table 54—exemplary gRNA sequences useful for targeting HELIOS.

The material submitted herewith in electronic (.txt) form and comprisingAppendices 1-47 (Tables 8-54, respectively) is incorporated herein byreference, specifically:

Appendix 1 (file name: Table8.txt; date created: May 9, 2016; file size:223,026 bytes);

Appendix 2 (file name: Table9.txt; date created: May 9, 2016; file size:327,895 bytes);

Appendix 3 (file name: Table10.txt; date created: May 9, 2016; filesize: 280,849 bytes);

Appendix 4 (file name: Table11.txt; date created: May 9, 2016; filesize: 998,046 bytes);

Appendix 5 (file name: Table12.txt; date created: May 9, 2016; file size4,717,823 bytes);

Appendix 6 (file name: Table13.txt; date created: May 9, 2016; file size2,813,407 bytes);

Appendix 7 (file name: Table14.txt; date created: May 9, 2016; file size6,568,742 bytes);

Appendix 8 (file name: Table15.txt; date created: May 9, 2016; file size728,685 bytes);

Appendix 9 (file name: Table16.txt; date created: May 9, 2016; file size766,106 bytes);

Appendix 10 (file name Table17.txt; date created: May 9, 2016; file size874,435 bytes);

Appendix 11 (file name Table18.txt; date created: May 9, 2016; file size232,536 bytes);

Appendix 12 (file name Table19.txt; date created: May 9, 2016; file size539,932 bytes);

Appendix 13 (file name Table20.txt; date created: May 9, 2016; file size2,256,084 bytes);

Appendix 14 (file name Table21.txt; date created: May 9, 2016; file size1,303,081 bytes);

Appendix 15 (file name Table22.txt; date created: May 9, 2016; file size6,821,299 bytes);

Appendix 16 (file name Table23.txt; date created: May 9, 2016; file size1,095,386 bytes);

Appendix 17 (file name Table24.txt; date created: May 9, 2016; file size941,281 bytes);

Appendix 18 (file name Table25.txt; date created: May 9, 2016; file size378,368 bytes);

Appendix 19 (file name Table26.txt; date created: May 9, 2016; file size2,706,509 bytes);

Appendix 20 (file name Table27.txt; date created: May 9, 2016; file size3,578,977 bytes);

Appendix 21 (file name Table28.txt; date created: May 9, 2016; file size3,638,039 bytes);

Appendix 22 (file name Table29.txt; date created: May 9, 2016; file size5,083,645 bytes);

Appendix 23 (file name Table30.txt; date created: May 9, 2016; file size4,481,092 bytes);

Appendix 24 (file name Table31.txt; date created: May 9, 2016; file size393,567 bytes);

Appendix 25 (file name Table32.txt; date created: May 9, 2016; file size675,503 bytes);

Appendix 26 (file name Table33.txt; date created: May 9, 2016; file size1,002,258 bytes);

Appendix 27 (file name Table34.txt; date created: May 9, 2016; file size454,540 bytes);

Appendix 28 (file name Table35.txt; date created: May 9, 2016; file size10,463,522 bytes);

Appendix 29 (file name Table36.txt; date created: May 9, 2016; file size2,755,910 bytes);

Appendix 30 (file name Table37.txt; date created: May 9, 2016; file size733,191 bytes);

Appendix 31 (file name Table38.txt; date created: May 9, 2016; file size1,674,331 bytes);

Appendix 32 (file name Table39.txt; date created: May 9, 2016; file size2,622,419 bytes);

Appendix 33 (file name Table40.txt; date created: May 9, 2016; file size2,049,613 bytes);

Appendix 34 (file name Table41.txt; date created: May 9, 2016; file size4,016,813 bytes);

Appendix 35 (file name Table42.txt; date created: May 9, 2016; file size4,177,884 bytes);

Appendix 36 (file name Table43.txt; date created: May 9, 2016; file size4,326,759 bytes);

Appendix 37 (file name Table44.txt; date created: May 9, 2016; file size1,007,674 bytes);

Appendix 38 (file name Table45.txt; date created: May 9, 2016; file size1,690,144 bytes);

Appendix 39 (file name Table46.txt; date created: May 9, 2016; file size5,427,706 bytes);

Appendix 40 (file name Table47.txt; date created: May 9, 2016; file size3,510,941 bytes);

Appendix 41 (file name Table48.txt; date created: May 9, 2016; file size3,043,135 bytes);

Appendix 42 (file name Table49.txt; date created: May 9, 2016; file size274,899,026 bytes);

Appendix 43 (file name Table50.txt; date created: May 9, 2016; file size7,273,026 bytes);

Appendix 44 (file name Table51.txt; date created: May 9, 2016; file size2,336,524 bytes);

Appendix 45 (file name Table52.txt; date created: May 9, 2016; file size274,899,026 bytes);

Appendix 46 (file name Table53.txt; date created: May 9, 2016; file size1,820,108 bytes); and

Appendix 47 (file name Table54.txt; date created: May 9, 2016; file size17,165,777 bytes).

DETAILED DESCRIPTION OF THE INVENTION

Recent advances in stem cell biology have made it possible tocontemplate the use of a subject's own cells as an unlimited source fortransplantation, as generally depicted, for example, in FIG. 1 .Unfortunately, genome editing and the generation of induced pluripotentstem cells (iPSCs) followed by the differentiation of such iPSCs remainsa costly, time consuming and highly variable process, with regards topluripotency, epigenetic status, capacity for differentiation, andgenomic stability. Moreover, changes occurring during genome editing andprolonged culturing have been found to trigger an adaptive immuneresponse, resulting in immune rejection of even autologous stemcell-derived transplants. To overcome the problem of a subject's immunerejection of stem cell-derived transplants, the present inventors havedeveloped and disclose herein a universal donor stem cell thatrepresents a viable source for any transplantable cell type.Advantageously, the universal stem cells disclosed herein are notrejected by the recipient subject's immune system, regardless of thesubject's genetic make-up.

The inventions disclosed herein employ genome editing technologies(e.g., the CRISPR/Cas or TALEN systems) to reduce or eliminateexpression of critical immune genes (e.g., by deleting genomic DNA ofcritical immune genes) or, in certain instances, inserttolerance-inducing factors, in human ES cells and iPSCs, rendering themand the differentiated cells prepared therefrom hypoimmunogenic and lessprone to immune rejection by a subject into which such cells aretransplanted. As used herein to characterize a cell, the term“hypoimmunogenic” generally means that such cell is less prone to immunerejection by a subject into which such cells are transplanted. Forexample, relative to an unaltered wild-type cell, such a hypoimmunogeniccell may be about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 97.5%, 99% or more less prone to immune rejection by a subject intowhich such cells are transplanted. In some aspects, genome editingtechnologies (e.g., the CRISPR/Cas or TALEN systems) are used tomodulate (e.g., reduce or eliminate) the expression of MHC-I and MHC-IIgenes.

In certain embodiments, the inventions disclosed herein relate to a stemcell, the genome of which has been altered to reduce or delete criticalcomponents of HLA expression. Similarly, in certain embodiments, theinventions disclosed herein relate to a stem cell, the genome of whichhas been altered to insert one or more tolerance inducing factors. Thepresent invention contemplates altering target polynucleotide sequencesin any manner which is available to the skilled artisan, for example,utilizing a TALEN or a CRISPR/Cas system. Such CRISPR/Cas systems canemploy a variety of Cas proteins (Haft et al. PLoS Comput Biol. 2005;1(6)e60). In some embodiments, the CRISPR/Cas system is a CRISPR type Isystem. In some embodiments, the CRISPR/Cas system is a CRISPR type IIsystem. In some embodiments, the CRISPR/Cas system is a CRISPR type Vsystem. It should be understood that although examples of methodsutilizing CRISPR/Cas (e.g., Cas9 and Cpf1) and TALEN are described indetail herein, the invention is not limited to the use of thesemethods/systems. Other methods of targeting polynucleotide sequences toreduce or ablate expression in target cells known to the skilled artisancan be utilized herein.

The present inventions contemplate altering, e.g., modifying orcleaving, target polynucleotide sequences in a cell for any purpose, butparticularly such that the expression or activity of the encoded productis reduced or eliminated. For example, CRISPR/Cas systems may be used totarget transcriptional regulators of antigen presentation to produce ahypoimmunogenic stem cell. In some embodiments, the targetpolynucleotide sequence in a cell (e.g., ES cells or iPSCs) is alteredto produce a mutant cell. As used herein, a “mutant cell” generallyrefers to a cell with a resulting genotype that differs from itsoriginal genotype or the wild-type cell. In some instances, a “mutantcell” exhibits a mutant phenotype, for example when a normallyfunctioning stem gene is altered using the CRISPR/Cas systems. In someembodiments, the target polynucleotide sequence in a cell is altered tocorrect or repair a genetic mutation (e.g., to restore a normalphenotype to the cell). In some embodiments, the target polynucleotidesequence in a cell is altered to induce a genetic mutation (e.g., todisrupt the function of a gene or genomic element).

In some embodiments, the alteration is an indel. As used herein, “indel”refers to a mutation resulting from an insertion, deletion, or acombination thereof. As will be appreciated by those skilled in the art,an indel in a coding region of a genomic sequence will result in aframeshift mutation, unless the length of the indel is a multiple ofthree. In some embodiments, the alteration is a point mutation. As usedherein, “point mutation” refers to a substitution that replaces one ofthe nucleotides. A CRISPR/Cas system can be used to induce an indel ofany length or a point mutation in a target polynucleotide sequence.

In some embodiments, the alteration results in a knock out of the targetpolynucleotide sequence or a portion thereof. For example, knocking outa target polynucleotide sequence in a cell can be performed in vitro, invivo or ex vivo for both therapeutic and research purposes. Knocking outa target polynucleotide sequence in a cell can be useful for treating orpreventing a disorder associated with expression of the targetpolynucleotide sequence (e.g., by knocking out a mutant allele in a cellex vivo and introducing those cells comprising the knocked out mutantallele into a subject).

As used herein, “knock out” includes deleting all or a portion of thetarget polynucleotide sequence in a way that interferes with thefunction of the target polynucleotide sequence or its expressionproduct.

In some embodiments, the alteration results in reduced expression of thetarget polynucleotide sequence. The terms “decrease,” “reduced,”“reduction,” and “decrease” are all used herein generally to mean adecrease by a statistically significant amount. However, for avoidanceof doubt, “decreased,” “reduced,” “reduction,” “decrease” includes adecrease by at least 10% as compared to a reference level, for example adecrease by at least about 20%, or at least about 30%, or at least about40%, or at least about 50%, or at least about 60%, or at least about70%, or at least about 80%, or at least about 90% or up to and includinga 100% decrease (i.e. absent level as compared to a reference sample),or any decrease between 10-100% as compared to a reference level.

The terms “increased,” “increase” or “enhance” or “activate” are allused herein to generally mean an increase by a statically significantamount; for the avoidance of any doubt, the terms “increased”,“increase” or “enhance” or “activate” means an increase of at least 10%as compared to a reference level, for example an increase of at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% increaseor any increase between 10-100% as compared to a reference level, or atleast about a 2-fold, or at least about a 3-fold, or at least about a4-fold, or at least about a 5-fold or at least about a 10-fold increase,or any increase between 2-fold and 10-fold or greater as compared to areference level.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) below normal, or lower, concentration of the marker. The termrefers to statistical evidence that there is a difference. It is definedas the probability of making a decision to reject the null hypothesiswhen the null hypothesis is actually true. The decision is often madeusing the p-value.

In some embodiments, the alteration is a homozygous alteration. In someembodiments, the alteration is a heterozygous alteration.

In some embodiments, the alteration results in correction of the targetpolynucleotide sequence from an undesired sequence to a desiredsequence. CRISPR/Cas systems can be used to correct any type of mutationor error in a target polynucleotide sequence. For example, CRISPR/Cassystems can be used to insert a nucleotide sequence that is missing froma target polynucleotide sequence due to a deletion. CRISPR/Cas systemscan also be used to delete or excise a nucleotide sequence from a targetpolynucleotide sequence due to an insertion mutation. In some instances,CRISPR/Cas systems can be used to replace an incorrect nucleotidesequence with a correct nucleotide sequence (e.g., to restore functionto a target polynucleotide sequence that is impaired due to a loss offunction mutation).

CRISPR/Cas systems can alter target polynucleotides with surprisinglyhigh efficiency. In certain embodiments, the efficiency of alteration isat least about 5%. In certain embodiments, the efficiency of alterationis at least about 10%. In certain embodiments, the efficiency ofalteration is from about 10% to about 80%. In certain embodiments, theefficiency of alteration is from about 30% to about 80%. In certainembodiments, the efficiency of alteration is from about 50% to about80%. In some embodiments, the efficiency of alteration is greater thanor equal to about 80%. In some embodiments, the efficiency of alterationis greater than or equal to about 85%. In some embodiments, theefficiency of alteration is greater than or equal to about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, or about 99%. In some embodiments, the efficiency ofalteration is equal to about 100%.

In some embodiments, the target polynucleotide sequence is a genomicsequence. In some embodiments, the target polynucleotide sequence is ahuman genomic sequence. In some embodiments, the target polynucleotidesequence is a mammalian genomic sequence. In some embodiments, thetarget polynucleotide sequence is a vertebrate genomic sequence.

In some embodiments, CRISPR/Cas systems include a Cas protein or anucleic acid sequence encoding the Cas protein and at least one to tworibonucleic acids (e.g., gRNAs) that are capable of directing the Casprotein to and hybridizing to a target motif of a target polynucleotidesequence. In some embodiments, CRISPR/Cas systems include a Cas proteinor a nucleic acid sequence encoding the Cas protein and a singleribonucleic acid or at least one pair of ribonucleic acids (e.g., gRNAs)that are capable of directing the Cas protein to and hybridizing to atarget motif of a target polynucleotide sequence. As used herein,“protein” and “polypeptide” are used interchangeably to refer to aseries of amino acid residues joined by peptide bonds (i.e., a polymerof amino acids) and include modified amino acids (e.g., phosphorylated,glycated, glycosolated, etc.) and amino acid analogs. Exemplarypolypeptides or proteins include gene products, naturally occurringproteins, homologs, paralogs, fragments and other equivalents, variants,and analogs of the above.

In some embodiments, a Cas protein comprises one or more amino acidsubstitutions or modifications. In some embodiments, the one or moreamino acid substitutions comprise a conservative amino acidsubstitution. In some instances, substitutions and/or modifications canprevent or reduce proteolytic degradation and/or extend the half-life ofthe polypeptide in a cell. In some embodiments, the Cas protein cancomprise a peptide bond replacement (e.g., urea, thiourea, carbamate,sulfonyl urea, etc.). In some embodiments, the Cas protein can comprisea naturally occurring amino acid. In some embodiments, the Cas proteincan comprise an alternative amino acid (e.g., D-amino acids, beta-aminoacids, homocysteine, phosphoserine, etc.). In some embodiments, a Casprotein can comprise a modification to include a moiety (e.g.,PEGylation, glycosylation, lipidation, acetylation, end-capping, etc.).

In some embodiments, a Cas protein comprises a core Cas protein.Exemplary Cas core proteins include, but are not limited to Cast, Cas2,Cas3, Cas4, Cas5, Cas6, Cas7, Cas8 and Cas9. In some embodiments, a Casprotein comprises a Cas protein of an E. coli subtype (also known asCASS2). Exemplary Cas proteins of the E. Coli subtype include, but arenot limited to Cse1, Cse2, Cse3, Cse4, and Cas5e. In some embodiments, aCas protein comprises a Cas protein of the Ypest subtype (also known asCASS3). Exemplary Cas proteins of the Ypest subtype include, but are notlimited to Csy1, Csy2, Csy3, and Csy4. In some embodiments, a Casprotein comprises a Cas protein of the Nmeni subtype (also known asCASS4). Exemplary Cas proteins of the Nmeni subtype include, but are notlimited to Csn1 and Csn2. In some embodiments, a Cas protein comprises aCas protein of the Dvulg subtype (also known as CASS1). Exemplary Casproteins of the Dvulg subtype include Csd1, Csd2, and Cas5d. In someembodiments, a Cas protein comprises a Cas protein of the Tneap subtype(also known as CASS7). Exemplary Cas proteins of the Tneap subtypeinclude, but are not limited to, Cst1, Cst2, Cas5t. In some embodiments,a Cas protein comprises a Cas protein of the Hmari subtype. ExemplaryCas proteins of the Hmari subtype include, but are not limited to Csh1,Csh2, and Cas5h. In some embodiments, a Cas protein comprises a Casprotein of the Apern subtype (also known as CASS5). Exemplary Casproteins of the Apern subtype include, but are not limited to Csa1,Csa2, Csa3, Csa4, Csa5, and Cas5a. In some embodiments, a Cas proteincomprises a Cas protein of the Mtube subtype (also known as CASS6).Exemplary Cas proteins of the Mtube subtype include, but are not limitedto Csm1, Csm2, Csm3, Csm4, and Csm5. In some embodiments, a Cas proteincomprises a RAMP module Cas protein. Exemplary RAMP module Cas proteinsinclude, but are not limited to, Cmr1, Cmr2, Cmr3, Cmr4, Cmr5, and Cmr6.

In some embodiments, the Cas protein is a Streptococcus pyogenes Cas9protein or a functional portion thereof. In some embodiments, the Casprotein is a Staphylococcus aureus Cas9 protein or a functional portionthereof. In some embodiments, the Cas protein is a Streptococcusthermophilus Cas9 protein or a functional portion thereof. In someembodiments, the Cas protein is a Neisseria meningitides Cas9 protein ora functional portion thereof. In some embodiments, the Cas protein is aTreponema denticola Cas9 protein or a functional portion thereof. Insome embodiments, the Cas protein is Cas9 protein from any bacterialspecies or functional portion thereof. Cas9 protein is a member of thetype II CRISPR systems which typically include a trans-coded small RNA(tracrRNA), endogenous ribonuclease 3 (mc) and a Cas protein. Cas 9protein (also known as CRISPR-associated endonuclease Cas9/Csn1) is apolypeptide comprising 1368 amino acids. An exemplary amino acidsequence of a Cas9 protein (SEQ ID NO: 1) is shown in FIG. 12 . Cas 9contains 2 endonuclease domains, including an RuvC-like domain (residues7-22, 759-766 and 982-989) which cleaves target DNA that isnoncomplementary to crRNA, and an HNH nuclease domain (residues 810-872)which cleave target DNA complementary to crRNA. In FIG. 12 , theRuvC-like domain is highlighted in yellow and the HNH nuclease domain isunderlined.

In some embodiments, the Cas protein is Cpf1 protein or a functionalportion thereof. In some embodiments, the Cas protein is Cpf1 from anybacterial species or functional portion thereof. In some aspects, Cpf1is a Francisella novicida U112 protein or a functional portion thereof.In some aspects, Cpf1 is a Acidaminococcus sp. BV3L6 protein or afunctional portion thereof. In some aspects, Cpf1 is a Lachnospiraceaebacterium ND2006 protein or a function portion thereof. Cpf1 protein isa member of the type V CRISPR systems. Cpf1 protein is a polypeptidecomprising about 1300 amino acids. Cpf1 contains a RuvC-likeendonuclease domain. Cpf1 cleaves target DNA in a staggered patternusing a single ribonuclease domain. The staggered DNA double-strandedbreak results in a 4 or 5-nt 5′ overhang.

As used herein, “functional portion” refers to a portion of a peptidewhich retains its ability to complex with at least one ribonucleic acid(e.g., guide RNA (gRNA)) and cleave a target polynucleotide sequence. Insome embodiments, the functional portion comprises a combination ofoperably linked Cas9 protein functional domains selected from the groupconsisting of a DNA binding domain, at least one RNA binding domain, ahelicase domain, and an endonuclease domain. In some embodiments, thefunctional portion comprises a combination of operably linked Cpf1protein functional domains selected from the group consisting of a DNAbinding domain, at least one RNA binding domain, a helicase domain, andan endonuclease domain. In some embodiments, the functional domains forma complex. In some embodiments, a functional portion of the Cas9 proteincomprises a functional portion of a RuvC-like domain. In someembodiments, a functional portion of the Cas9 protein comprises afunctional portion of the HNH nuclease domain. In some embodiments, afunctional portion of the Cpf1 protein comprises a functional portion ofa RuvC-like domain.

It should be appreciated that the present invention contemplates variousways of contacting a target polynucleotide sequence with a Cas protein(e.g., Cas9). In some embodiments, exogenous Cas protein can beintroduced into the cell in polypeptide form. In certain embodiments,Cas proteins can be conjugated to or fused to a cell-penetratingpolypeptide or cell-penetrating peptide. As used herein,“cell-penetrating polypeptide” and “cell-penetrating peptide” refers toa polypeptide or peptide, respectively, which facilitates the uptake ofmolecule into a cell. The cell-penetrating polypeptides can contain adetectable label.

In certain embodiments, Cas proteins can be conjugated to or fused to acharged protein (e.g., that carries a positive, negative or overallneutral electric charge). Such linkage may be covalent. In someembodiments, the Cas protein can be fused to a superpositively chargedGFP to significantly increase the ability of the Cas protein topenetrate a cell (Cronican et al. ACS Chem Biol. 2010; 5(8):747-52). Incertain embodiments, the Cas protein can be fused to a proteintransduction domain (PTD) to facilitate its entry into a cell. ExemplaryPTDs include Tat, oligoarginine, and penetratin. In some embodiments,the Cas9 protein comprises a Cas9 polypeptide fused to acell-penetrating peptide. In some embodiments, the Cas9 proteincomprises a Cas9 polypeptide fused to a PTD. In some embodiments, theCas9 protein comprises a Cas9 polypeptide fused to a tat domain. In someembodiments, the Cas9 protein comprises a Cas9 polypeptide fused to anoligoarginine domain. In some embodiments, the Cas9 protein comprises aCas9 polypeptide fused to a penetratin domain. In some embodiments, theCas9 protein comprises a Cas9 polypeptide fused to a superpositivelycharged GFP. In some embodiments, the Cpf1 protein comprises a Cpf1polypeptide fused to a cell-penetrating peptide. In some embodiments,the Cpf1 protein comprises a Cpf1 polypeptide fused to a PTD. In someembodiments, the Cpf1 protein comprises a Cpf1 polypeptide fused to atat domain. In some embodiments, the Cpf1 protein comprises a Cpf1polypeptide fused to an oligoarginine domain. In some embodiments, theCpf1 protein comprises a Cpf1 polypeptide fused to a penetratin domain.In some embodiments, the Cpf1 protein comprises a Cpf1 polypeptide fusedto a superpositively charged GFP.

In some embodiments, the Cas protein can be introduced into a cellcontaining the target polynucleotide sequence in the form of a nucleicacid encoding the Cas protein (e.g., Cas9 or Cpf1). The process ofintroducing the nucleic acids into cells can be achieved by any suitabletechnique. Suitable techniques include calcium phosphate orlipid-mediated transfection, electroporation, and transduction orinfection using a viral vector. In some embodiments, the nucleic acidcomprises DNA. In some embodiments, the nucleic acid comprises amodified DNA, as described herein. In some embodiments, the nucleic acidcomprises mRNA. In some embodiments, the nucleic acid comprises amodified mRNA, as described herein (e.g., a synthetic, modified mRNA).

In some embodiments, nucleic acids encoding Cas protein and nucleicacids encoding the at least one to two ribonucleic acids are introducedinto a cell via viral transduction (e.g., lentiviral transduction).

In some embodiments, the Cas protein is complexed with one to tworibonucleic acids. In some embodiments, the Cas protein is complexedwith two ribonucleic acids. In some embodiments, the Cas protein iscomplexed with one ribonucleic acid. In some embodiments, the Casprotein is encoded by a modified nucleic acid, as described herein(e.g., a synthetic, modified mRNA).

The methods of the present invention contemplate the use of anyribonucleic acid that is capable of directing a Cas protein to andhybridizing to a target motif of a target polynucleotide sequence. Insome embodiments, at least one of the ribonucleic acids comprisestracrRNA. In some embodiments, at least one of the ribonucleic acidscomprises CRISPR RNA (crRNA). In some embodiments, a single ribonucleicacid comprises a guide RNA that directs the Cas protein to andhybridizes to a target motif of the target polynucleotide sequence in acell. In some embodiments, at least one of the ribonucleic acidscomprises a guide RNA that directs the Cas protein to and hybridizes toa target motif of the target polynucleotide sequence in a cell. In someembodiments, both of the one to two ribonucleic acids comprise a guideRNA that directs the Cas protein to and hybridizes to a target motif ofthe target polynucleotide sequence in a cell. The ribonucleic acids ofthe present invention can be selected to hybridize to a variety ofdifferent target motifs, depending on the particular CRISPR/Cas systememployed, and the sequence of the target polynucleotide, as will beappreciated by those skilled in the art. The one to two ribonucleicacids can also be selected to minimize hybridization with nucleic acidsequences other than the target polynucleotide sequence. In someembodiments, the one to two ribonucleic acids hybridize to a targetmotif that contains at least two mismatches when compared with all othergenomic nucleotide sequences in the cell. In some embodiments, the oneto two ribonucleic acids hybridize to a target motif that contains atleast one mismatch when compared with all other genomic nucleotidesequences in the cell. In some embodiments, the one to two ribonucleicacids are designed to hybridize to a target motif immediately adjacentto a deoxyribonucleic acid motif recognized by the Cas protein. In someembodiments, each of the one to two ribonucleic acids are designed tohybridize to target motifs immediately adjacent to deoxyribonucleic acidmotifs recognized by the Cas protein which flank a mutant allele locatedbetween the target motifs.

In some embodiments, at least one of the one to two ribonucleic acidscomprises a sequence selected from the group consisting of theribonucleic acid sequences of SEQ ID NOs: 2-817976. In some embodiments,at least one ribonucleic acid comprises a sequence selected from thegroup consisting of the ribonucleic acid sequences of SEQ ID NOs:2-817976.

In some embodiments, at least one of the one to two ribonucleic acidscomprises a sequence with a single nucleotide mismatch to a sequenceselected from the group consisting of the ribonucleic acid sequences ofSEQ ID NOs: 2-817976. In some embodiments, at least one ribonucleic acidcomprises a sequence with a single nucleotide mismatch to a sequenceselected from the group consisting of the ribonucleic acid sequences ofSEQ ID NOs: 2-817976.

In some embodiments, each of the one to two ribonucleic acids comprisesguide RNAs that directs the Cas protein to and hybridizes to a targetmotif of the target polynucleotide sequence in a cell. In someembodiments, one or two ribonucleic acids (e.g., guide RNAs) arecomplementary to and/or hybridize to sequences on the same strand of atarget polynucleotide sequence. In some embodiments, one or tworibonucleic acids (e.g., guide RNAs) are complementary to and/orhybridize to sequences on the opposite strands of a targetpolynucleotide sequence. In some embodiments, the one or two ribonucleicacids (e.g., guide RNAs) are not complementary to and/or do nothybridize to sequences on the opposite strands of a targetpolynucleotide sequence. In some embodiments, the one or two ribonucleicacids (e.g., guide RNAs) are complementary to and/or hybridize tooverlapping target motifs of a target polynucleotide sequence. In someembodiments, the one or two ribonucleic acids (e.g., guide RNAs) arecomplementary to and/or hybridize to offset target motifs of a targetpolynucleotide sequence.

In some embodiments, the target motif is a 17 to 23 nucleotide DNAsequence. In some embodiments, the target motif is at least 20nucleotides in length. In some embodiments, the target motif is a20-nucleotide DNA sequence. In some embodiments, the target motif is a17 to 23-nucleotide DNA sequence and immediately precedes an NRG motif.In some aspects, the NRG motif is NGG or NAG. In some embodiments, thetarget motif is a 20-nucleotide DNA sequence and immediately precedes anNGG motif recognized by the Cas protein. In some embodiments, the targetmotif is a 20-nucleotide DNA sequence and immediately precedes an NAGmotif recognized by the Cas protein. In some embodiments, the targetmotif is a 20-nucleotide DNA sequence beginning with G and immediatelyprecedes an NGG motif recognized by the Cas protein. In someembodiments, the target motif is G(N)19NGG. In some embodiments, thetarget motif is (N)20NGG.

In some embodiments, the target motif is a 17 to 23-nucleotide DNAsequence and immediately precedes an NNGRRT motif. In some embodiments,the target motif is a 20 nucleotide DNA sequence and immediatelyprecedes an NNGRRT motif. In some embodiments, the target motif is a 17to 23-nucleotide DNA sequence and immediately precedes an NNNRRT motif.In some embodiments, the target motif is a 20 nucleotide DNA sequenceand immediately precedes an NNNRRT motif. In some embodiments, thetarget motif is a 17 to 23-nucleotide DNA sequence and immediatelyprecedes an NNAGAAW motif. In some embodiments, the target motif is a 20nucleotide DNA sequence and immediately precedes an NNAGAAW motif. Insome embodiments, the target motif is a 17 to 23-nucleotide DNA sequenceand immediately precedes an NNNNGATT motif. In some embodiments, thetarget motif is a 20 nucleotide DNA sequence and immediately precedes anNNNNGATT motif. In some embodiments, the target motif is a 17 to23-nucleotide DNA sequence and immediately precedes an NAAAAC motif. Insome embodiments, the target motif is a 20 nucleotide DNA sequence andimmediately precedes an NAAAAC motif. In some embodiments, the targetmotif is a 17 to 23-nucleotide DNA sequence having a 5′ T-rich region(e.g., TTTN motif). In some embodiments, the target motif is a 20nucleotide DNA sequence having a 5′ T-rich region (e.g., TTTN motif).

In some embodiments, the target motif is a 17 to 23-nucleotide DNAsequence and immediately precedes an NRG motif (e.g., NGG or NAG)recognized by a S. pyogenes Cas9 protein. In some embodiments, thetarget motif is a 17 to 23-nucleotide DNA sequence and immediatelyprecedes an NNGRRT motif recognized by a S. aureus Cas9 protein. In someembodiments, the target motif is a 17 to 23-nucleotide DNA sequence andimmediately precedes an NNNRRT motif recognized by a S. aureus Cas9protein. In some embodiments, the target motif is a 17 to 23-nucleotideDNA sequence and immediately precedes an NNAGAAW motif recognized by aS. thermophilus Cas9 protein. In some embodiments, the target motif is a17 to 23-nucleotide DNA sequence and immediately precedes an NNNNGATTmotif recognized by N. meningitides Cas9 protein. In some embodiments,the target motif is a 17 to 23-nucleotide DNA sequence and immediatelyprecedes an NAAAAC motif recognized by T. denticola Cas9 protein. Insome embodiments, the target motif is a 17 to 23-nucleotide DNA sequencehaving a 5′ T-rich region (e.g., TTTN motif) recognized byAcidaminococcus or Lachnospiraceae Cpf1 protein.

In some embodiments, the one to two ribonucleic acids hybridize to atarget motif that contains at least two mismatches when compared withall other genomic nucleotide sequences in the cell. In some embodiments,the one to two ribonucleic acids hybridize to a target motif thatcontains at least one mismatch when compared with all other genomicnucleotide sequences in the cell. Those skilled in the art willappreciate that a variety of techniques can be used to select suitabletarget motifs for minimizing off-target effects (e.g., bioinformaticsanalyses). In some embodiments, the one to two ribonucleic acids aredesigned to hybridize to a target motif immediately adjacent to adeoxyribonucleic acid motif recognized by the Cas protein. In someembodiments, each of the one to two ribonucleic acids are designed tohybridize to target motifs immediately adjacent to deoxyribonucleic acidmotifs recognized by the Cas protein which flank a mutant allele locatedbetween the target motifs.

In some aspects, the target polynucleotide sequence in a cell is alteredto reduce or eliminate expression and/or activity of one or morecritical immune genes in the cell using a genetic editing system (e.g.,TALENs, CRISPR/Cas, etc.). In some embodiments, the present disclosureprovides that the target polynucleotide sequence in a cell is altered todelete a contiguous stretch of genomic DNA (e.g., delete one or morecritical immune genes) from one or both alleles of the cell (e.g., usinga CRISPR/Cas system). In some embodiments, the target polynucleotidesequence in a cell is altered to insert a genetic mutation in one orboth alleles of the cell (e.g., using a CRISPR/Cas system). In stillother embodiments, the universal stem cells disclosed herein may besubject to complementary genome editing approaches (e.g., using aCRISPR/Cas system), whereby such stem cells are modified to both deletecontiguous stretches of genomic DNA (e.g., critical immune genes) fromone or both alleles of the cell, as well as to insert one or moretolerance-inducing factors, such as HLA-G or PD-L1, into one or bothalleles of the cells to locally suppress the immune system and improvetransplant engraftment.

The universal stem cells disclosed herein may be differentiated intorelevant cell types to assess HLA expression, as well as the evaluationof immunogenicity of the universal stem cell lines, for example, in apre-clinical humanized mouse model. For example, the universal stemcells disclosed herein may be incubated under appropriate conditions anddifferentiated into mesenchymal progenitors cells (MPCs),hypoimmunogenic cardiomyocytes, endothelial cells (ECs), macrophages,hepatocytes, beta cells (e.g., pancreatic beta cells), or neuralprogenitor cells (NPCs).

The universal stem cells and methods disclosed herein will have anenormous impact on regenerative medicine by leading the way torigorously tested universal donor stem cell lines that could be grown upand differentiated into a very large numbers of cells, made widelyavailable to all medical institutions, and used on demand to treatpatients suffering from degenerative illnesses, and thereby make itunnecessary to use a patient's own cells on a case-by-case basis as asource for autologous transplantation. Moreover, as the resulting cellproducts will be protected from immune attack, they will represent a newform of treatment for autoimmune diseases such as MS (multiplesclerosis) and diabetes, where autologous cells would still be prone toimmune attack. Immunoprivileged universal donor stem cell-derived cellproducts, however, will be protected from autoimmune rejection.

The universal stem cells disclosed herein may be used, for example, todiagnose, monitor, treat and/or cure the presence or progression of adisease or condition in a subject. As used herein, a “subject” means ahuman or animal. In certain embodiments, the subject is a human. Incertain embodiments, the subject is an adolescent. In certainembodiments, the subject is treated in vivo, in vitro and/or in utero.In certain aspects, a subject in need of treatment in accordance withthe methods disclosed herein has a condition or is suspected or atincreased risk of developing such condition.

As depicted in FIG. 2A, HLA represents an immunologic barrier to thesuccessful transplantation of stem cells or differentiated stem cells ina subject. Disclosed herein are novel compositions, cells and relatedmethods that are useful for overcoming the HLA immunologic barrier totransplantation. As illustrated in FIG. 2B, major histocompatibilitycomplex (MHC) is a locus on human Chr. 6p21, which encodes a highlypolymorphic gene family of surface molecules that define donorcompatibility during organ transplantation. MHC class I (MHC-I) and MHCclass II (MHC-II) play essential roles in the activation of adaptiveimmune responses by presenting antigens to T lymphocytes. A comparisonof the MHC-II and MHC-I enhanceosomes is provided in FIG. 13 . Humanshave three classical MHC-Ia molecules (HLA-A, HLA-B, and HLA-C), whichare vital to the detection and elimination of viruses, cancerous cells,and transplanted cells. In addition, there are three non-classicalMHC-Ib molecules (HLA-E, HLA-F, and HLA-G), which have immune regulatoryfunctions. While MHC's serve a vital cellular function, in certaincontexts, such as cell-based transplantation therapies, they may alsocontribute to immune rejection. Provided herein are novel cells,compositions and methods that are useful for addressing such HLA-basedimmune rejection of transplanted cells.

Knock-Outs

In certain aspects, the inventions disclosed herein relate to genomicmodifications of one or more targeted polynucleotide sequences of thestem cell genome that regulates the expression of MHC-I and/or MHC-II.In some aspects, a genetic editing system is used to modify one or moretargeted polynucleotide sequences. In some aspects, a CRISPR/Cas systemis used to delete the one or more targeted polynucleotide sequences.MHC-I molecules are composed of MHC-encoded heavy chains and theinvariant subunit β2-microglobulin (B2M). Antigen-derived peptides arepresented by MHC-I-B2M complexes at the cell surface to CD8 T cellscarrying an antigen-specific T cell receptor. Peptides are mostlyproduced from the degradation of cytoplasmic proteins by a specializedproteasome or immunoproteasome, which is optimized to generate MHC classI peptides and contains several IFN-γ-inducible subunits. Unlike MHC-II,which is found mainly in antigen-presenting cells, MHC-Ia isubiquitously expressed in almost all nucleated cells (Pamer, et al.,Annu Rev Immunol (1998) 16:323-358.). Both MHC-I and MHC-II genes arehighly inducible by IFN-γ stimulation.

The efficient removal of the HLA barrier can be accomplished by one ormore of the following: (1) targeting the polymorphic HLA alleles (HLA-A,-B, -C) and MHC-II genes directly; (2) removal of B2M, which willprevent surface trafficking of all MHC-I molecules; and/or (3) deletionof components of the MHC enhanceosomes, such as NLRC5, RFX-5, -ANK, and-AP, IRF1, NF-Y, and CIITA that are critical for HLA expression.

In certain embodiments, HLA expression is interfered with. In someaspects, HLA expression is interfered with by targeting individual HLAs(e.g., knocking out expression of HLA-A, HLA-B and/or HLA-C), targetingtranscriptional regulators of HLA expression (e.g., knocking outexpression of NLRC5, CIITA, RFX5, RFXAP, RFXANK, NFY-A, NFY-B, NFY-Cand/or IRF-1), blocking surface trafficking of MHC class I molecules(e.g., knocking out expression of B2M and/or TAP1), and/or targetingHLA-Razor.

In certain aspects, the stem cells disclosed herein do not express oneor more human leukocyte antigens (e.g., HLA-A, HLA-B and/or HLA-C)corresponding to MHC-I and/or MHC-II and are thus characterized as beinghypoimmunogenic. For example, in certain aspects, the stem cellsdisclosed herein have been modified such that the stem cell or adifferentiated stem cell prepared therefrom do not express or exhibitreduced expression of one or more of the following MHC-I molecules:HLA-A, HLA-B and HLA-C. In some aspects, one or more of HLA-A, HLA-B andHLA-C may be “knocked-out” of a cell. A cell that has a knocked-outHLA-A gene, HLA-B gene, and/or HLA-C gene may exhibit reduced oreliminated expression of each knocked-out gene. See FIGS. 13A-J.

In some aspects, the present disclosure provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof comprising a genome inwhich the HLA-A gene has been edited to delete a contiguous stretch ofgenomic DNA, thereby reducing or eliminating surface expression of MHCclass I molecules in the cell or population thereof. The contiguousstretch of genomic DNA can be deleted by contacting the cell orpopulation thereof with a Cas protein or a nucleic acid encoding the Casprotein and at least one ribonucleic acid or at least one pair ofribonucleic acids selected from the group consisting of SEQ ID NOs:2-1418.

In certain aspects, the present disclosure provides a method foraltering a target HLA-A sequence in a cell comprising contacting theHLA-A sequence with a clustered regularly interspaced short palindromicrepeats-associated (Cas) protein and at least one ribonucleic acid or atleast one pair of ribonucleic acids, wherein the ribonucleic acidsdirect Cas protein to and hybridize to a target motif of the targetHLA-A polynucleotide sequence, wherein the target HLA-A polynucleotidesequence is cleaved, and wherein the at least one ribonucleic acid orthe at least one pair of ribonucleic acids is selected from the groupconsisting of SEQ ID NOs: 2-1418.

In some aspects, the present disclosure provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof comprising a genome inwhich the HLA-B gene has been edited to delete a contiguous stretch ofgenomic DNA, thereby reducing or eliminating surface expression of MHCclass I molecules in the cell or population thereof. The contiguousstretch of genomic DNA can be deleted by contacting the cell orpopulation thereof with a Cas protein or a nucleic acid encoding the Casprotein and at least one ribonucleic acid or at least one pair ofribonucleic acids selected from the group consisting of SEQ ID NOs:1419-3277.

In certain aspects, the present disclosure provides a method foraltering a target HLA-B sequence in a cell comprising contacting theHLA-B sequence with a clustered regularly interspaced short palindromicrepeats-associated (Cas) protein and at least one ribonucleic acid or atleast one pair of ribonucleic acids, wherein the ribonucleic acidsdirect Cas protein to and hybridize to a target motif of the targetHLA-B polynucleotide sequence, wherein the target HLA-B polynucleotidesequence is cleaved, and wherein the at least one ribonucleic acid orthe at least one pair of ribonucleic acids is selected from the groupconsisting of SEQ ID NOs: 1419-3277.

In some aspects, the present disclosure provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof comprising a genome inwhich the HLA-C gene has been edited to delete a contiguous stretch ofgenomic DNA, thereby reducing or eliminating surface expression of MHCclass I molecules in the cell or population thereof. The contiguousstretch of genomic DNA can be deleted by contacting the cell orpopulation thereof with a Cas protein or a nucleic acid encoding the Casprotein and at least one ribonucleic acid or at least one pair ofribonucleic acids selected from the group consisting of SEQ ID NOs:3278-5183.

In certain aspects, the present disclosure provides a method foraltering a target HLA-C sequence in a cell comprising contacting theHLA-C sequence with a clustered regularly interspaced short palindromicrepeats-associated (Cas) protein and at least one ribonucleic acid or atleast one pair of ribonucleic acids, wherein the ribonucleic acidsdirect Cas protein to and hybridize to a target motif of the targetHLA-C polynucleotide sequence, wherein the target HLA-C polynucleotidesequence is cleaved, and wherein the at least one ribonucleic acid orthe at least one pair of ribonucleic acids is selected from the groupconsisting of SEQ ID NOs: 3278-5183.

In certain embodiments, the expression of MHC-I or MHC-II is modulatedby targeting and deleting a contiguous stretch of genomic DNA therebyreducing or eliminating expression of a target gene, for example, NLRC5.As used herein, the term “modulate” is used consistently with its use inthe art, i.e., meaning to cause or facilitate a qualitative orquantitative change, alteration, or modification in a process, pathway,or phenomenon of interest. Without limitation, such change may be anincrease, decrease, or change in relative strength or activity ofdifferent components or branches of the process, pathway, or phenomenon.In certain aspects, the target gene is NLRC5, CIITA, RFX5, RFXAP,RFXANK, NFY-A, NFY-B, NFY-C or IRF-1.

In certain aspects, the inventions disclosed herein modulate (e.g.,reduce or eliminate) the expression of MHC-II genes by targeting andmodulating (e.g., reducing or eliminating) Class II transactivator(CIITA) expression. In some aspects, the modulation occurs using aCRISPR/Cas system. CIITA is a member of the NLR or nucleotide bindingdomain (NBD) leucine-rich repeat (LRR) family of proteins and regulatesthe transcription of MHC-II by associating with the MHC enhanceosome.The expression of CIITA is induced in B cells and dendritic cells as afunction of developmental stage and is inducible by IFN-γ in most celltypes. Aside from CIITA, NLR proteins are localized in the cytoplasm andcontribute to the innate immune response by recognizing microbialproducts and exogenous danger signals, leading to inflammation and/orcell death.

In some aspects, the present disclosure provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof comprising a genome inwhich the Class II transactivator (CIITA) gene has been edited to deletea contiguous stretch of genomic DNA, thereby reducing or eliminatingsurface expression of MHC class II molecules in the cell or populationthereof.

The contiguous stretch of genomic DNA can be deleted by contacting thecell or population thereof with a Cas protein or a nucleic acid encodingthe Cas protein and at least one ribonucleic acid or at least one pairof ribonucleic acids selected from the group consisting of SEQ ID NOs:5184-36352.

The present invention contemplates genomically editing human cells tocleave CIITA gene sequences, as well as editing the genome of such cellsto alter one or more additional target polynucleotide sequences (e.g.,NLRC5 and/or B2M). It should be appreciated that cleaving a CIITAgenomic sequence using one or more gRNAs or gRNA pairs described hereinand a Cas protein could result in partial or complete deletion of thetarget CIITA genomic sequence, depending on the number of gRNAs or gRNApairs selected, as well as their targets.

In some aspects, the invention provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof, each cell comprising amodified genome comprising a genomic modification in which the CIITAgene has been edited to delete a contiguous stretch of genomic DNA,thereby reducing or eliminating MHC Class II molecule surface expressionand/or activity in the cell. In some embodiments, the contiguous stretchof genomic DNA has been deleted by contacting the cell with a Casprotein or a nucleic acid encoding the Cas protein and a pair ofribonucleic acids having sequences selected from the group consisting ofSEQ ID NOs: 5184-36352.

In some aspects, the invention provides a method for altering a targetCIITA polynucleotide sequence in a cell comprising contacting the CIITApolynucleotide sequence with a clustered regularly interspaced shortpalindromic repeats-associated (Cas) protein and from one to tworibonucleic acids, wherein the ribonucleic acids direct Cas protein toand hybridize to a target motif of the target CIITA polynucleotidesequence, wherein the target CIITA polynucleotide sequence is cleaved,and wherein at least one of the one to two ribonucleic acids areselected from the group consisting of SEQ ID NOs: 5184-36352.

In some aspects, the invention provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof, each cell comprising amodified genome comprising: a genomic modification in which the CIITAgene has been edited to reduce or eliminate CIITA surface expressionand/or activity in the cell by contacting the cell with a Cas protein ora nucleic acid encoding the Cas protein and at least one ribonucleicacid having a sequence selected from the group consisting of SEQ ID NOs:5184-36352.

In some aspects, the invention provides a method for altering a targetCIITA polynucleotide sequence in a cell comprising contacting the CIITApolynucleotide sequence with a clustered regularly interspaced shortpalindromic repeats-associated (Cas) protein and at least oneribonucleic acid, wherein the ribonucleic acid directs Cas protein toand hybridizes to a target motif of the target CIITA polynucleotidesequence, wherein the target CIITA polynucleotide sequence is cleaved,and wherein the at least one ribonucleic acid is selected from the groupconsisting of SEQ ID NOs: 5184-36352.

In certain aspects, the inventions disclosed herein modulate (e.g.,reduce or eliminate) the expression of MHC-I genes by targeting andmodulating (e.g., reducing or eliminating) expression of the NLR family,CARD domain containing 5/NOD27/CLR16.1 (NLRC5). In some aspects, themodulation occurs using a CRISPR/Cas system. NLRC5 is a criticalregulator of MHC-I-mediated immune responses and, similar to CIITA,NLRC5 is highly inducible by IFN-γ and can translocate into the nucleus.NLRC5 activates the promoters of MHC-I genes and induces thetranscription of MHC-I as well as related genes involved in MHC-Iantigen presentation.

In some aspects, the present disclosure provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof comprising a genome inwhich the NLRC5 gene has been edited to delete a contiguous stretch ofgenomic DNA, thereby reducing or eliminating surface expression of MHCclass I molecules in the cell or population thereof.

The contiguous stretch of genomic DNA can be deleted by contacting thecell or population thereof with a Cas protein or a nucleic acid encodingthe Cas protein and at least one ribonucleic acid or at least one pairof ribonucleic acids selected from the group consisting of SEQ ID NOs:36353-81239.

The present invention contemplates genomically editing human cells tocleave NLRC5 gene sequences, as well as editing the genome of such cellsto alter one or more additional target polynucleotide sequences (e.g.,CIITA and/or B2M). It should be appreciated that cleaving a NLRC5genomic sequence using one or more gRNAs or gRNA pairs described hereinand a Cas protein could result in partial or complete deletion of thetarget NLRC5 genomic sequence, depending on the number of gRNAs or gRNApairs selected, as well as their targets.

In some aspects, the invention provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof, each cell comprising amodified genome comprising a genomic modification in which the NLRC5gene has been edited to delete a contiguous stretch of genomic DNA,thereby reducing or eliminating MHC Class I molecule surface expressionand/or activity in the cell. In some embodiments, the contiguous stretchof genomic DNA has been deleted by contacting the cell with a Casprotein or a nucleic acid encoding the Cas protein and a pair ofribonucleic acids having sequences selected from the group consisting ofSEQ ID NOs: 36353-81239.

In some aspects, the invention provides a method for altering a targetNLRC5 polynucleotide sequence in a cell comprising contacting the NLRC5polynucleotide sequence with a clustered regularly interspaced shortpalindromic repeats-associated (Cas) protein and from one to tworibonucleic acids, wherein the ribonucleic acids direct Cas protein toand hybridize to a target motif of the target NLRC5 polynucleotidesequence, wherein the target NLRC5 polynucleotide sequence is cleaved,and wherein at least one of the one to two ribonucleic acids areselected from the group consisting of SEQ ID NOs: 36353-81239.

In some aspects, the invention provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof, each cell comprising amodified genome comprising: a genomic modification in which the NLRC5gene has been edited to reduce or eliminate MHC Class I molecule surfaceexpression and/or activity in the cell by contacting the cell with a Casprotein or a nucleic acid encoding the Cas protein and at least oneribonucleic acid having a sequence selected from the group consisting ofSEQ ID NOs: 36353-81239.

In some aspects, the invention provides a method for altering a targetNLRC5 polynucleotide sequence in a cell comprising contacting the NLRC5polynucleotide sequence with a clustered regularly interspaced shortpalindromic repeats-associated (Cas) protein and at least oneribonucleic acid, wherein the ribonucleic acid directs Cas protein toand hybridizes to a target motif of the target NLRC5 polynucleotidesequence, wherein the target NLRC5 polynucleotide sequence is cleaved,and wherein the at least one ribonucleic acid is selected from the groupconsisting of SEQ ID NOs: 36353-81239.

In certain embodiments, the inventions disclosed herein modulate (e.g.,reduce or eliminate) the expression of MHC-I genes by targeting andmodulating (e.g., reducing or eliminating) expression of the accessorychain B2M. In some aspects, the modulation occurs using a CRISPR/Cassystem. By modulating (e.g., reducing or deleting) expression of B2M,surface trafficking of MHC-I molecules is blocked and the cell renderedhypoimmunogenic.

In some aspects, the present disclosure provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof comprising a genome inwhich the β2-microglobulin (B2M) gene has been edited to delete acontiguous stretch of genomic DNA, thereby reducing or eliminatingsurface expression of MHC class I molecules in the cell or populationthereof.

The contiguous stretch of genomic DNA can be deleted by contacting thecell or population thereof with a Cas protein or a nucleic acid encodingthe Cas protein and at least one ribonucleic acid or at least one pairof ribonucleic acids selected from the group consisting of SEQ ID NOs:81240-85644.

The present invention contemplates genomically editing human cells tocleave B2M gene sequences, as well as editing the genome of such cellsto alter one or more additional target polynucleotide sequences (e.g.,NLRC5 and/or CIITA). It should be appreciated that cleaving a B2Mgenomic sequence using one or more gRNAs or gRNA pairs described hereinand a Cas protein could result in partial or complete deletion of thetarget B2M genomic sequence, depending on the number of gRNAs or gRNApairs selected, as well as their targets.

In some aspects, the invention provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof, each cell comprising amodified genome comprising a genomic modification in which the B2M genehas been edited to delete a contiguous stretch of genomic DNA, therebyreducing or eliminating MHC Class I molecule surface expression and/oractivity in the cell. In some embodiments, the contiguous stretch ofgenomic DNA has been deleted by contacting the cell with a Cas proteinor a nucleic acid encoding the Cas protein and a pair of ribonucleicacids having sequences selected from the group consisting of SEQ ID NOs:81240-85644.

In some aspects, the invention provides a method for altering a targetB2M polynucleotide sequence in a cell comprising contacting the B2Mpolynucleotide sequence with a clustered regularly interspaced shortpalindromic repeats-associated (Cas) protein and from one to tworibonucleic acids, wherein the ribonucleic acids direct Cas protein toand hybridize to a target motif of the target B2M polynucleotidesequence, wherein the target B2M polynucleotide sequence is cleaved, andwherein at least one of the one to two ribonucleic acids are selectedfrom the group consisting of SEQ ID NOs: 81240-85644.

In some aspects, the invention provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof, each cell comprising amodified genome comprising: a genomic modification in which the B2M genehas been edited to reduce or eliminate MHC Class I molecule surfaceexpression and/or activity in the cell by contacting the cell with a Casprotein or a nucleic acid encoding the Cas protein and at least oneribonucleic acid having a sequence selected from the group consisting ofSEQ ID NOs: 81240-85644.

In some aspects, the invention provides a method for altering a targetB2M polynucleotide sequence in a cell comprising contacting the B2Mpolynucleotide sequence with a clustered regularly interspaced shortpalindromic repeats-associated (Cas) protein and at least oneribonucleic acid, wherein the ribonucleic acid directs Cas protein toand hybridizes to a target motif of the target B2M polynucleotidesequence, wherein the target B2M polynucleotide sequence is cleaved, andwherein the at least one ribonucleic acid is selected from the groupconsisting of SEQ ID NOs: 81240-85644.

In certain aspects, the inventions disclosed herein modulate (e.g.,reduce or eliminate) the expression of MHC-I genes by targeting andmodulating (e.g., reducing or eliminating) expression of one or more ofRFX5. In some aspects, the modulation occurs using a CRISPR/Cas system.

In some aspects, the present disclosure provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof comprising a genome inwhich the RFX5 gene has been edited to delete a contiguous stretch ofgenomic DNA, thereby reducing or eliminating surface expression of MHCclass I molecules in the cell or population thereof.

The contiguous stretch of genomic DNA can be deleted by contacting thecell or population thereof with a Cas protein or a nucleic acid encodingthe Cas protein and at least one ribonucleic acid or at least one pairof ribonucleic acids selected from the group consisting of SEQ ID NOs:85645-90115.

The present invention contemplates genomically editing human cells tocleave RFX5 gene sequences, as well as editing the genome of such cellsto alter one or more additional target polynucleotide sequences. Itshould be appreciated that cleaving a RFX5 genomic sequence using one ormore gRNAs or gRNA pairs described herein and a Cas protein could resultin partial or complete deletion of the target RFX5 genomic sequence,depending on the number of gRNAs or gRNA pairs selected, as well astheir targets.

In some aspects, the invention provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof, each cell comprising amodified genome comprising a genomic modification in which the RFX5 genehas been edited to delete a contiguous stretch of genomic DNA, therebyreducing or eliminating MHC Class I molecule surface expression and/oractivity in the cell. In some embodiments, the contiguous stretch ofgenomic DNA has been deleted by contacting the cell with a Cas proteinor a nucleic acid encoding the Cas protein and a pair of ribonucleicacids having sequences selected from the group consisting of SEQ ID NOs:85645-90115.

In some aspects, the invention provides a method for altering a targetRFX5 polynucleotide sequence in a cell comprising contacting the RFX5polynucleotide sequence with a clustered regularly interspaced shortpalindromic repeats-associated (Cas) protein and from one to tworibonucleic acids, wherein the ribonucleic acids direct Cas protein toand hybridize to a target motif of the target RFX5 polynucleotidesequence, wherein the target RFX5 polynucleotide sequence is cleaved,and wherein at least one of the one to two ribonucleic acids areselected from the group consisting of SEQ ID NOs: 85645-90115.

In some aspects, the invention provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof, each cell comprising amodified genome comprising: a genomic modification in which the RFX5gene has been edited to reduce or eliminate MHC Class I molecule surfaceexpression and/or activity in the cell by contacting the cell with a Casprotein or a nucleic acid encoding the Cas protein and at least oneribonucleic acid having a sequence selected from the group consisting ofSEQ ID NOs: 85645-90115.

In some aspects, the invention provides a method for altering a targetRFX5 polynucleotide sequence in a cell comprising contacting the RFX5polynucleotide sequence with a clustered regularly interspaced shortpalindromic repeats-associated (Cas) protein and at least oneribonucleic acid, wherein the ribonucleic acid directs Cas protein toand hybridizes to a target motif of the target RFX5 polynucleotidesequence, wherein the target RFX5 polynucleotide sequence is cleaved,and wherein the at least one ribonucleic acid is selected from the groupconsisting of SEQ ID NOs: 85645-90115.

In certain aspects, the inventions disclosed herein modulate (e.g.,reduce or eliminate) the expression of MHC-I genes by targeting andmodulating (e.g., reducing or eliminating) expression of one or more ofRFXAP. In some aspects, the modulation occurs using a CRISPR/Cas system.

In some aspects, the present disclosure provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof comprising a genome inwhich the RFXAP gene has been edited to delete a contiguous stretch ofgenomic DNA, thereby reducing or eliminating surface expression of MHCclass I molecules in the cell or population thereof.

The contiguous stretch of genomic DNA can be deleted by contacting thecell or population thereof with a Cas protein or a nucleic acid encodingthe Cas protein and at least one ribonucleic acid or at least one pairof ribonucleic acids selected from the group consisting of SEQ ID NOs:90116-95635.

The present invention contemplates genomically editing human cells tocleave RFXAP gene sequences, as well as editing the genome of such cellsto alter one or more additional target polynucleotide sequences. Itshould be appreciated that cleaving a RFXAP genomic sequence using oneor more gRNAs or gRNA pairs described herein and a Cas protein couldresult in partial or complete deletion of the target RFXAP genomicsequence, depending on the number of gRNAs or gRNA pairs selected, aswell as their targets.

In some aspects, the invention provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof, each cell comprising amodified genome comprising a genomic modification in which the RFXAPgene has been edited to delete a contiguous stretch of genomic DNA,thereby reducing or eliminating MHC Class I molecule surface expressionand/or activity in the cell. In some embodiments, the contiguous stretchof genomic DNA has been deleted by contacting the cell with a Casprotein or a nucleic acid encoding the Cas protein and a pair ofribonucleic acids having sequences selected from the group consisting ofSEQ ID NOs: 90116-95635.

In some aspects, the invention provides a method for altering a targetRFXAP polynucleotide sequence in a cell comprising contacting the RFXAPpolynucleotide sequence with a clustered regularly interspaced shortpalindromic repeats-associated (Cas) protein and from one to tworibonucleic acids, wherein the ribonucleic acids direct Cas protein toand hybridize to a target motif of the target RFXAP polynucleotidesequence, wherein the target RFXAP polynucleotide sequence is cleaved,and wherein at least one of the one to two ribonucleic acids areselected from the group consisting of SEQ ID NOs: 90116-95635.

In some aspects, the invention provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof, each cell comprising amodified genome comprising: a genomic modification in which the RFXAPgene has been edited to reduce or eliminate MHC Class I molecule surfaceexpression and/or activity in the cell by contacting the cell with a Casprotein or a nucleic acid encoding the Cas protein and at least oneribonucleic acid having a sequence selected from the group consisting ofSEQ ID NOs: 90116-95635.

In some aspects, the invention provides a method for altering a targetRFXAP polynucleotide sequence in a cell comprising contacting the RFXAPpolynucleotide sequence with a clustered regularly interspaced shortpalindromic repeats-associated (Cas) protein and at least oneribonucleic acid, wherein the ribonucleic acid directs Cas protein toand hybridizes to a target motif of the target RFXAP polynucleotidesequence, wherein the target RFXAP polynucleotide sequence is cleaved,and wherein the at least one ribonucleic acid is selected from the groupconsisting of SEQ ID NOs: 90116-95635.

In certain aspects, the inventions disclosed herein modulate (e.g.,reduce or eliminate) the expression of MHC-I genes by targeting andmodulating (e.g., reducing or eliminating) expression of one or more ofRFXANK. In some aspects, the modulation occurs using a CRISPR/Cassystem.

In some aspects, the present disclosure provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof comprising a genome inwhich the RFXANK gene has been edited to delete a contiguous stretch ofgenomic DNA, thereby reducing or eliminating surface expression of MHCclass I molecules in the cell or population thereof.

The contiguous stretch of genomic DNA can be deleted by contacting thecell or population thereof with a Cas protein or a nucleic acid encodingthe Cas protein and at least one ribonucleic acid or at least one pairof ribonucleic acids selected from the group consisting of SEQ ID NOs:95636-102318.

The present invention contemplates genomically editing human cells tocleave RFXANK gene sequences, as well as editing the genome of suchcells to alter one or more additional target polynucleotide sequences.It should be appreciated that cleaving a RFXANK genomic sequence usingone or more gRNAs or gRNA pairs described herein and a Cas protein couldresult in partial or complete deletion of the target RFXANK genomicsequence, depending on the number of gRNAs or gRNA pairs selected, aswell as their targets.

In some aspects, the invention provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof, each cell comprising amodified genome comprising a genomic modification in which the RFXAPgene has been edited to delete a contiguous stretch of genomic DNA,thereby reducing or eliminating MHC Class I molecule surface expressionand/or activity in the cell. In some embodiments, the contiguous stretchof genomic DNA has been deleted by contacting the cell with a Casprotein or a nucleic acid encoding the Cas protein and a pair ofribonucleic acids having sequences selected from the group consisting ofSEQ ID NOs: 95636-102318.

In some aspects, the invention provides a method for altering a targetRFXANK polynucleotide sequence in a cell comprising contacting theRFXANK polynucleotide sequence with a clustered regularly interspacedshort palindromic repeats-associated (Cas) protein and from one to tworibonucleic acids, wherein the ribonucleic acids direct Cas protein toand hybridize to a target motif of the target RFXANK polynucleotidesequence, wherein the target RFXANK polynucleotide sequence is cleaved,and wherein at least one of the one to two ribonucleic acids areselected from the group consisting of SEQ ID NOs: 95636-102318.

In some aspects, the invention provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof, each cell comprising amodified genome comprising: a genomic modification in which the RFXANKgene has been edited to reduce or eliminate MHC Class I molecule surfaceexpression and/or activity in the cell by contacting the cell with a Casprotein or a nucleic acid encoding the Cas protein and at least oneribonucleic acid having a sequence selected from the group consisting ofSEQ ID NOs: 95636-102318.

In some aspects, the invention provides a method for altering a targetRFXANK polynucleotide sequence in a cell comprising contacting theRFXANK polynucleotide sequence with a clustered regularly interspacedshort palindromic repeats-associated (Cas) protein and at least oneribonucleic acid, wherein the ribonucleic acid directs Cas protein toand hybridizes to a target motif of the target RFXANK polynucleotidesequence, wherein the target RFXANK polynucleotide sequence is cleaved,and wherein the at least one ribonucleic acid is selected from the groupconsisting of SEQ ID NOs: 95636-102318.

In certain aspects, the inventions disclosed herein modulate (e.g.,reduce or eliminate) the expression of MHC-I genes by targeting andmodulating (e.g., reducing or eliminating) expression of one or more ofNFY-A. In some aspects, the modulation occurs using a CRISPR/Cas system.

In some aspects, the present disclosure provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof comprising a genome inwhich the NFY-A gene has been edited to delete a contiguous stretch ofgenomic DNA, thereby reducing or eliminating surface expression of MHCclass I molecules in the cell or population thereof.

The contiguous stretch of genomic DNA can be deleted by contacting thecell or population thereof with a Cas protein or a nucleic acid encodingthe Cas protein and at least one ribonucleic acid or at least one pairof ribonucleic acids selected from the group consisting of SEQ ID NOs:102319-121796.

The present invention contemplates genomically editing human cells tocleave NFY-A gene sequences, as well as editing the genome of such cellsto alter one or more additional target polynucleotide sequences. Itshould be appreciated that cleaving a NFY-A genomic sequence using oneor more gRNAs or gRNA pairs described herein and a Cas protein couldresult in partial or complete deletion of the target NFY-A genomicsequence, depending on the number of gRNAs or gRNA pairs selected, aswell as their targets.

In some aspects, the invention provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof, each cell comprising amodified genome comprising a genomic modification in which the NFY-Agene has been edited to delete a contiguous stretch of genomic DNA,thereby reducing or eliminating MHC Class I molecule surface expressionand/or activity in the cell. In some embodiments, the contiguous stretchof genomic DNA has been deleted by contacting the cell with a Casprotein or a nucleic acid encoding the Cas protein and a pair ofribonucleic acids having sequences selected from the group consisting ofSEQ ID NOs: 102319-121796.

In some aspects, the invention provides a method for altering a targetNFY-A polynucleotide sequence in a cell comprising contacting the NFY-Apolynucleotide sequence with a clustered regularly interspaced shortpalindromic repeats-associated (Cas) protein and from one to tworibonucleic acids, wherein the ribonucleic acids direct Cas protein toand hybridize to a target motif of the target NFY-A polynucleotidesequence, wherein the target NFY-A polynucleotide sequence is cleaved,and wherein at least one of the one to two ribonucleic acids areselected from the group consisting of SEQ ID NOs: 102319-121796.

In some aspects, the invention provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof, each cell comprising amodified genome comprising: a genomic modification in which the NFY-Agene has been edited to reduce or eliminate MHC Class I molecule surfaceexpression and/or activity in the cell by contacting the cell with a Casprotein or a nucleic acid encoding the Cas protein and at least oneribonucleic acid having a sequence selected from the group consisting ofSEQ ID NOs: 102319-121796.

In some aspects, the invention provides a method for altering a targetNFY-A polynucleotide sequence in a cell comprising contacting the NFY-Apolynucleotide sequence with a clustered regularly interspaced shortpalindromic repeats-associated (Cas) protein and at least oneribonucleic acid, wherein the ribonucleic acid directs Cas protein toand hybridizes to a target motif of the target NFY-A polynucleotidesequence, wherein the target NFY-A polynucleotide sequence is cleaved,and wherein the at least one ribonucleic acid is selected from the groupconsisting of SEQ ID NOs: 102319-121796.

In certain aspects, the inventions disclosed herein modulate (e.g.,reduce or eliminate) the expression of MHC-I genes by targeting andmodulating (e.g., reducing or eliminating) expression of one or more ofNFY-B. In some aspects, the modulation occurs using a CRISPR/Cas system.

In some aspects, the present disclosure provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof comprising a genome inwhich the NFY-B gene has been edited to delete a contiguous stretch ofgenomic DNA, thereby reducing or eliminating surface expression of MHCclass I molecules in the cell or population thereof.

The contiguous stretch of genomic DNA can be deleted by contacting thecell or population thereof with a Cas protein or a nucleic acid encodingthe Cas protein and at least one ribonucleic acid or at least one pairof ribonucleic acids selected from the group consisting of SEQ ID NOs:121797-135112.

The present invention contemplates genomically editing human cells tocleave NFY-B gene sequences, as well as editing the genome of such cellsto alter one or more additional target polynucleotide sequences. Itshould be appreciated that cleaving a NFY-B genomic sequence using oneor more gRNAs or gRNA pairs described herein and a Cas protein couldresult in partial or complete deletion of the target NFY-B genomicsequence, depending on the number of gRNAs or gRNA pairs selected, aswell as their targets.

In some aspects, the invention provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof, each cell comprising amodified genome comprising a genomic modification in which the NFY-Bgene has been edited to delete a contiguous stretch of genomic DNA,thereby reducing or eliminating MHC Class I molecule surface expressionand/or activity in the cell. In some embodiments, the contiguous stretchof genomic DNA has been deleted by contacting the cell with a Casprotein or a nucleic acid encoding the Cas protein and a pair ofribonucleic acids having sequences selected from the group consisting ofSEQ ID NOs: 121797-135112.

In some aspects, the invention provides a method for altering a targetNFY-B polynucleotide sequence in a cell comprising contacting the NFY-Bpolynucleotide sequence with a clustered regularly interspaced shortpalindromic repeats-associated (Cas) protein and from one to tworibonucleic acids, wherein the ribonucleic acids direct Cas protein toand hybridize to a target motif of the target NFY-B polynucleotidesequence, wherein the target NFY-B polynucleotide sequence is cleaved,and wherein at least one of the one to two ribonucleic acids areselected from the group consisting of SEQ ID NOs: 121797-135112.

In some aspects, the invention provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof, each cell comprising amodified genome comprising: a genomic modification in which the NFY-Bgene has been edited to reduce or eliminate MHC Class I molecule surfaceexpression and/or activity in the cell by contacting the cell with a Casprotein or a nucleic acid encoding the Cas protein and at least oneribonucleic acid having a sequence selected from the group consisting ofSEQ ID NOs: 121797-135112.

In some aspects, the invention provides a method for altering a targetNFY-B polynucleotide sequence in a cell comprising contacting the NFY-Bpolynucleotide sequence with a clustered regularly interspaced shortpalindromic repeats-associated (Cas) protein and at least oneribonucleic acid, wherein the ribonucleic acid directs Cas protein toand hybridizes to a target motif of the target NFY-B polynucleotidesequence, wherein the target NFY-B polynucleotide sequence is cleaved,and wherein the at least one ribonucleic acid is selected from the groupconsisting of SEQ ID NOs: 121797-135112.

In certain aspects, the inventions disclosed herein modulate (e.g.,reduce or eliminate) the expression of MHC-I genes by targeting andmodulating (e.g., reducing or eliminating) expression of one or more ofNFY-C. In some aspects, the modulation occurs using a CRISPR/Cas system.

In some aspects, the present disclosure provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof comprising a genome inwhich the NFY-C gene has been edited to delete a contiguous stretch ofgenomic DNA, thereby reducing or eliminating surface expression of MHCclass I molecules in the cell or population thereof.

The contiguous stretch of genomic DNA can be deleted by contacting thecell or population thereof with a Cas protein or a nucleic acid encodingthe Cas protein and at least one ribonucleic acid or at least one pairof ribonucleic acids selected from the group consisting of SEQ ID NOs:135113-176601.

The present invention contemplates genomically editing human cells tocleave NFY-C gene sequences, as well as editing the genome of such cellsto alter one or more additional target polynucleotide sequences. Itshould be appreciated that cleaving a NFY-C genomic sequence using oneor more gRNAs or gRNA pairs described herein and a Cas protein couldresult in partial or complete deletion of the target NFY-C genomicsequence, depending on the number of gRNAs or gRNA pairs selected, aswell as their targets.

In some aspects, the invention provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof, each cell comprising amodified genome comprising a genomic modification in which the NFY-Cgene has been edited to delete a contiguous stretch of genomic DNA,thereby reducing or eliminating MHC Class I molecule surface expressionand/or activity in the cell. In some embodiments, the contiguous stretchof genomic DNA has been deleted by contacting the cell with a Casprotein or a nucleic acid encoding the Cas protein and a pair ofribonucleic acids having sequences selected from the group consisting ofSEQ ID NOs: 135113-176601.

In some aspects, the invention provides a method for altering a targetNFY-C polynucleotide sequence in a cell comprising contacting the NFY-Cpolynucleotide sequence with a clustered regularly interspaced shortpalindromic repeats-associated (Cas) protein and from one to tworibonucleic acids, wherein the ribonucleic acids direct Cas protein toand hybridize to a target motif of the target NFY-C polynucleotidesequence, wherein the target NFY-C polynucleotide sequence is cleaved,and wherein at least one of the one to two ribonucleic acids areselected from the group consisting of SEQ ID NOs: 135113-176601.

In some aspects, the invention provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof, each cell comprising amodified genome comprising: a genomic modification in which the NFY-Cgene has been edited to reduce or eliminate MHC Class I molecule surfaceexpression and/or activity in the cell by contacting the cell with a Casprotein or a nucleic acid encoding the Cas protein and at least oneribonucleic acid having a sequence selected from the group consisting ofSEQ ID NOs: 135113-176601.

In some aspects, the invention provides a method for altering a targetNFY-C polynucleotide sequence in a cell comprising contacting the NFY-Cpolynucleotide sequence with a clustered regularly interspaced shortpalindromic repeats-associated (Cas) protein and at least oneribonucleic acid, wherein the ribonucleic acid directs Cas protein toand hybridizes to a target motif of the target NFY-C polynucleotidesequence, wherein the target NFY-C polynucleotide sequence is cleaved,and wherein the at least one ribonucleic acid is selected from the groupconsisting of SEQ ID NOs: 135113-176601.

In certain aspects, the inventions disclosed herein modulate (e.g.,reduce or eliminate) the expression of MHC-I genes by targeting andmodulating (e.g., reducing or eliminating) expression of one or more ofIRF-1. In some aspects, the modulation occurs using a CRISPR/Cas system.

In some aspects, the present disclosure provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof comprising a genome inwhich the IRF-1 gene has been edited to delete a contiguous stretch ofgenomic DNA, thereby reducing or eliminating surface expression of MHCclass I molecules in the cell or population thereof.

The contiguous stretch of genomic DNA can be deleted by contacting thecell or population thereof with a Cas protein or a nucleic acid encodingthe Cas protein and at least one ribonucleic acid or at least one pairof ribonucleic acids selected from the group consisting of SEQ ID NOs:176602-182813.

The present invention contemplates genomically editing human cells tocleave IRF-1 gene sequences, as well as editing the genome of such cellsto alter one or more additional target polynucleotide sequences. Itshould be appreciated that cleaving a IRF-1 genomic sequence using oneor more gRNAs or gRNA pairs described herein and a Cas protein couldresult in partial or complete deletion of the target IRF-1 genomicsequence, depending on the number of gRNAs or gRNA pairs selected, aswell as their targets.

In some aspects, the invention provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof, each cell comprising amodified genome comprising a genomic modification in which the IRF-1gene has been edited to delete a contiguous stretch of genomic DNA,thereby reducing or eliminating MHC Class I molecule surface expressionand/or activity in the cell. In some embodiments, the contiguous stretchof genomic DNA has been deleted by contacting the cell with a Casprotein or a nucleic acid encoding the Cas protein and a pair ofribonucleic acids having sequences selected from the group consisting ofSEQ ID NOs: 176602-182813.

In some aspects, the invention provides a method for altering a targetIRF-1 polynucleotide sequence in a cell comprising contacting the IRF-1polynucleotide sequence with a clustered regularly interspaced shortpalindromic repeats-associated (Cas) protein and from one to tworibonucleic acids, wherein the ribonucleic acids direct Cas protein toand hybridize to a target motif of the target IRF-1 polynucleotidesequence, wherein the target IRF-1 polynucleotide sequence is cleaved,and wherein at least one of the one to two ribonucleic acids areselected from the group consisting of SEQ ID NOs: 176602-182813.

In some aspects, the invention provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof, each cell comprising amodified genome comprising: a genomic modification in which the IRF-1gene has been edited to reduce or eliminate MHC Class I molecule surfaceexpression and/or activity in the cell by contacting the cell with a Casprotein or a nucleic acid encoding the Cas protein and at least oneribonucleic acid having a sequence selected from the group consisting ofSEQ ID NOs: 176602-182813.

In some aspects, the invention provides a method for altering a targetIRF-1 polynucleotide sequence in a cell comprising contacting the IRF-1polynucleotide sequence with a clustered regularly interspaced shortpalindromic repeats-associated (Cas) protein and at least oneribonucleic acid, wherein the ribonucleic acid directs Cas protein toand hybridizes to a target motif of the target IRF-1 polynucleotidesequence, wherein the target IRF-1 polynucleotide sequence is cleaved,and wherein the at least one ribonucleic acid is selected from the groupconsisting of SEQ ID NOs: 176602-182813.

In some aspects, the present disclosure provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof comprising a genome inwhich the TAP1 gene has been edited to delete a contiguous stretch ofgenomic DNA, thereby reducing or eliminating surface expression of MHCclass I molecules in the cell or population thereof.

The contiguous stretch of genomic DNA can be deleted by contacting thecell or population thereof with a Cas protein or a nucleic acid encodingthe Cas protein and at least one ribonucleic acid or at least one pairof ribonucleic acids selected from the group consisting of SEQ ID NOs:182814-188371.

The present invention contemplates genomically editing human cells tocleave TAP1 gene sequences, as well as editing the genome of such cellsto alter one or more additional target polynucleotide sequences. Itshould be appreciated that cleaving a TAP1 genomic sequence using one ormore gRNAs or gRNA pairs described herein and a Cas protein could resultin partial or complete deletion of the target TAP1 genomic sequence,depending on the number of gRNAs or gRNA pairs selected, as well astheir targets.

In some aspects, the invention provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof, each cell comprising amodified genome comprising a genomic modification in which the TAP1 genehas been edited to delete a contiguous stretch of genomic DNA, therebyreducing or eliminating MHC Class I molecule surface expression and/oractivity in the cell. In some embodiments, the contiguous stretch ofgenomic DNA has been deleted by contacting the cell with a Cas proteinor a nucleic acid encoding the Cas protein and a pair of ribonucleicacids having sequences selected from the group consisting of SEQ ID NOs:182814-188371.

In some aspects, the invention provides a method for altering a targetTAP1 polynucleotide sequence in a cell comprising contacting the TAP1polynucleotide sequence with a clustered regularly interspaced shortpalindromic repeats-associated (Cas) protein and from one to tworibonucleic acids, wherein the ribonucleic acids direct Cas protein toand hybridize to a target motif of the target TAP1 polynucleotidesequence, wherein the target TAP1 polynucleotide sequence is cleaved,and wherein at least one of the one to two ribonucleic acids areselected from the group consisting of SEQ ID NOs: 182814-188371.

In some aspects, the invention provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof, each cell comprising amodified genome comprising: a genomic modification in which the TAP1gene has been edited to reduce or eliminate MHC Class I molecule surfaceexpression and/or activity in the cell by contacting the cell with a Casprotein or a nucleic acid encoding the Cas protein and at least oneribonucleic acid having a sequence selected from the group consisting ofSEQ ID NOs: 182814-188371.

In some aspects, the invention provides a method for altering a targetTAP1 polynucleotide sequence in a cell comprising contacting the TAP1polynucleotide sequence with a clustered regularly interspaced shortpalindromic repeats-associated (Cas) protein and at least oneribonucleic acid, wherein the ribonucleic acid directs Cas protein toand hybridizes to a target motif of the target TAP1 polynucleotidesequence, wherein the target TAP1 polynucleotide sequence is cleaved,and wherein the at least one ribonucleic acid is selected from the groupconsisting of SEQ ID NOs: 182814-188371.

In certain embodiments, gRNAs that allow simultaneous deletion of allMHC class I alleles by targeting a conserved region in the HLA genes areidentified as HLA Razors. In some aspects, the gRNAs are part of aCRISPR system. In alternative aspects, the gRNAs are part of a TALENsystem. In one aspect, an HLA Razor targeting an identified conservedregion in HLAs is depicted in FIG. 21A. In other aspects, multiple HLARazors targeting identified conserved regions are utilized. It isgenerally understood that any guide that targets a conserved region inHLAs can act as an HLA Razor.

Knock-In

In certain embodiments, tolerogenic factors can be inserted orreinserted into genome-edited stem cell lines to createimmune-privileged universal donor stem cell lines. In certainembodiments, the universal stem cells disclosed herein have been furthermodified to express one or more tolerogenic factors. Exemplarytolerogenic factors include, without limitation, one or more of HLA-C,HLA-E, HLA-F, HLA-G, PD-L1, CTLA-4-Ig, CD47, C1-inhibitor, and IL-35.

The present inventors have used genome editing systems, such as theCRISPR/Cas system, to facilitate the insertion of tolerogenic factors,such as the tolerogenic factors shown in Table 2 below, into a safeharbor locus, such as the AAVS1 locus, to actively inhibit immunerejection. As evidenced in FIGS. 11A-11C, the present inventors havesuccessfully expressed tolerogenic factors, such as PD-L1 and HLA-G,from a safe harbor locus.

TABLE 2 Tolerogenic factors that can be (re)introduced into genomeedited stem cell lines to create immune-privileged universal donor stemcell lines. Gene Receptor Target Cell HLA-G* KIR2DL4 NK cells HLA-C*KIR2DS1/L1 NK cells HLA-E* NKG2A/C NK cells PD-L1 PD-1 T cells CTLA4-IgCD28 APC/T cells CD47 SIRPα Macrophages C1-inhibitor Complement IL-35IL35R T reg *in the form of a peptide-B2M-HLA fusion construct, when(re)introduced into a B2M^(−/−) cell line

In some aspects, the present disclosure provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof comprising a genome inwhich the stem cell genome has been modified to express HLA-G. In someaspects, the present disclosure provides a method for altering a stemcell genome to express HLA-G. In certain aspects at least oneribonucleic acid or at least one pair of ribonucleic acids may beutilized to facilitate the insertion of HLA-G into a stem cell line. Incertain embodiments, the at least one ribonucleic acid or the at leastone pair of ribonucleic acids is selected from the group consisting ofSEQ ID NOs: 188372-189858.

In some aspects, the present disclosure provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof comprising a genome inwhich the stem cell genome has been modified to express HLA-C. In someaspects, the present disclosure provides a method for altering a stemcell genome to express HLA-C. In certain aspects at least oneribonucleic acid or at least one pair of ribonucleic acids may beutilized to facilitate the insertion of HLA-C into a stem cell line. Incertain embodiments, the at least one ribonucleic acid or the at leastone pair of ribonucleic acids is selected from the group consisting ofSEQ ID NOs: 3278-5183.

In some aspects, the present disclosure provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof comprising a genome inwhich the stem cell genome has been modified to express HLA-E. In someaspects, the present disclosure provides a method for altering a stemcell genome to express HLA-E. In certain aspects at least oneribonucleic acid or at least one pair of ribonucleic acids may beutilized to facilitate the insertion of HLA-E into a stem cell line. Incertain embodiments, the at least one ribonucleic acid or the at leastone pair of ribonucleic acids is selected from the group consisting ofSEQ ID NOs: 189859-193183.

In some aspects, the present disclosure provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof comprising a genome inwhich the stem cell genome has been modified to express PD-L1. In someaspects, the present disclosure provides a method for altering a stemcell genome to express PD-L1. In certain aspects at least oneribonucleic acid or at least one pair of ribonucleic acids may beutilized to facilitate the insertion of PD-L1 into a stem cell line. Incertain embodiments, the at least one ribonucleic acid or the at leastone pair of ribonucleic acids is selected from the group consisting ofSEQ ID NOs: 193184-200783.

In some aspects, the present disclosure provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof comprising a genome inwhich the stem cell genome has been modified to express CD-47. In someaspects, the present disclosure provides a method for altering a stemcell genome to express CD-47. In certain aspects at least oneribonucleic acid or at least one pair of ribonucleic acids may beutilized to facilitate the insertion of CD-47 into a stem cell line. Incertain embodiments, the at least one ribonucleic acid or the at leastone pair of ribonucleic acids is selected from the group consisting ofSEQ ID NOs: 200784-231885.

In some aspects, the present disclosure provides a stem cell (e.g.,hypoimmunogenic stem cell) or population thereof comprising a genome inwhich the stem cell genome has been modified to express HLA-F. In someaspects, the present disclosure provides a method for altering a stemcell genome to express HLA-F. In certain aspects at least oneribonucleic acid or at least one pair of ribonucleic acids may beutilized to facilitate the insertion of HLA-F into a stem cell line. Incertain embodiments, the at least one ribonucleic acid or the at leastone pair of ribonucleic acids is selected from the group consisting ofSEQ ID NOs: 688808-399754.

Other Target Modifications

In some embodiments, additional targets can be modified and/or deletedin universal cells, such as universal T cells, to improve their functionand/or tailor them to a specific therapeutic approach.

In some aspects, genes encoding for co-stimulatory molecules/receptorsthat engage cytotoxic T cells can be deleted by genome editing (Table3). Deletion of co-stimulatory molecules/receptors may occur to preventautoimmunity. In other aspects, genes encoding for co-inhibitorymolecules/receptors can be deleted by genome editing (Table 4). Deletionof co-inhibitory molecules/receptors may occur to prevent T cellinhibition by cancer cells, and may be useful in T cell-based cancerimmunotherapy.

TABLE 3 Co-stimulatory molecules that will be deleted to preventautoimmunity (e.g., to block interaction of transplanted cells with Tcells). Ligand on Cancer cells Receptor on T cells Ox40 L Ox40 GITRLGITR 4-1BBL 4-1BB CD58 CD2 B7-1, -2 CD28 B2-2 ICOS CD70 CD27 LIGHT HVEMSLAM SLAM CD155, CD112 CD226

TABLE 4 Co-inhibitory molecules/receptors that will be deleted toprevent T cell inhibition by cancer cells. Useful in T cell-based cancerimmunotherapy Ligand on Cancer cells Receptor on T cells B7/H1PD-L1 PD1B7-1/B7-2 CTLA4 MHC LAG3 CD155, CD112, CD113 TIGIT Galectin 9 TIM3 B7-1B7-H1/PD-L1 PD-L2 PD-1 B7-H3 TLT-2 CD153  CD30 VISTA ? HVEM CD160 HVEMBTLA Collagen LAIRI CD48 2B4/CD244

In some aspects, the present disclosure provides a stem cell orpopulation thereof comprising a genome in which the OX40 gene has beenedited to modify (e.g., delete) a contiguous stretch of genomic DNA. Incertain aspects, the present disclosure provides a method for altering atarget OX40 sequence in a cell. The contiguous stretch of genomic DNAcan be modified (e.g., deleted) by contacting the cell or populationthereof with a Cas protein or a nucleic acid encoding the Cas proteinand at least one ribonucleic acid or at least one pair of ribonucleicacids selected from the group consisting of SEQ ID NOs: 231886-234210.

In some aspects, the present disclosure provides a stem cell orpopulation thereof comprising a genome in which the GITR gene has beenedited to modify a contiguous stretch of genomic DNA. In certainaspects, the present disclosure provides a method for altering a targetGITR sequence in a cell. The contiguous stretch of genomic DNA can bemodified by contacting the cell or population thereof with a Cas proteinor a nucleic acid encoding the Cas protein and at least one ribonucleicacid or at least one pair of ribonucleic acids selected from the groupconsisting of SEQ ID NOs: 234211-236445.

In some aspects, the present disclosure provides a stem cell orpopulation thereof comprising a genome in which the 4-1BB gene has beenedited to modify a contiguous stretch of genomic DNA. In certainaspects, the present disclosure provides a method for altering a target4-1BB sequence in a cell. The contiguous stretch of genomic DNA can bemodified by contacting the cell or population thereof with a Cas proteinor a nucleic acid encoding the Cas protein and at least one ribonucleicacid or at least one pair of ribonucleic acids selected from the groupconsisting of SEQ ID NOs: 234211-236445.

In some aspects, the present disclosure provides a stem cell orpopulation thereof comprising a genome in which the CD28 gene has beenedited to modify a contiguous stretch of genomic DNA. In certainaspects, the present disclosure provides a method for altering a targetCD28 sequence in a cell. The contiguous stretch of genomic DNA can bemodified by contacting the cell or population thereof with a Cas proteinor a nucleic acid encoding the Cas protein and at least one ribonucleicacid or at least one pair of ribonucleic acids selected from the groupconsisting of SEQ ID NOs: 252807-274181.

In some aspects, the present disclosure provides a stem cell orpopulation thereof comprising a genome in which the B7-1 gene has beenedited to modify a contiguous stretch of genomic DNA. In certainaspects, the present disclosure provides a method for altering a targetB7-1 sequence in a cell. The contiguous stretch of genomic DNA can bemodified by contacting the cell or population thereof with a Cas proteinor a nucleic acid encoding the Cas protein and at least one ribonucleicacid or at least one pair of ribonucleic acids selected from the groupconsisting of SEQ ID NOs: 274182-295529.

In some aspects, the present disclosure provides a stem cell orpopulation thereof comprising a genome in which the B7-2 gene has beenedited to modify a contiguous stretch of genomic DNA. In certainaspects, the present disclosure provides a method for altering a targetB7-2 sequence in a cell. The contiguous stretch of genomic DNA can bemodified by contacting the cell or population thereof with a Cas proteinor a nucleic acid encoding the Cas protein and at least one ribonucleicacid or at least one pair of ribonucleic acids selected from the groupconsisting of SEQ ID NOs: 295530-324177.

In some aspects, the present disclosure provides a stem cell orpopulation thereof comprising a genome in which the ICOS gene has beenedited to modify a contiguous stretch of genomic DNA. In certainaspects, the present disclosure provides a method for altering a targetICOS sequence in a cell. The contiguous stretch of genomic DNA can bemodified by contacting the cell or population thereof with a Cas proteinor a nucleic acid encoding the Cas protein and at least one ribonucleicacid or at least one pair of ribonucleic acids selected from the groupconsisting of SEQ ID NOs: 324178-339974.

In some aspects, the present disclosure provides a stem cell orpopulation thereof comprising a genome in which the CD27 gene has beenedited to modify a contiguous stretch of genomic DNA. In certainaspects, the present disclosure provides a method for altering a targetCD27 sequence in a cell. The contiguous stretch of genomic DNA can bemodified by contacting the cell or population thereof with a Cas proteinor a nucleic acid encoding the Cas protein and at least one ribonucleicacid or at least one pair of ribonucleic acids selected from the groupconsisting of SEQ ID NOs: 339975-344266.

In some aspects, the present disclosure provides a stem cell orpopulation thereof comprising a genome in which the HVEM gene has beenedited to modify a contiguous stretch of genomic DNA. In certainaspects, the present disclosure provides a method for altering a targetHVEM sequence in a cell. The contiguous stretch of genomic DNA can bemodified by contacting the cell or population thereof with a Cas proteinor a nucleic acid encoding the Cas protein and at least one ribonucleicacid or at least one pair of ribonucleic acids selected from the groupconsisting of SEQ ID NOs: 344267-350722.

In some aspects, the present disclosure provides a stem cell orpopulation thereof comprising a genome in which the SLAM gene has beenedited to modify a contiguous stretch of genomic DNA. In certainaspects, the present disclosure provides a method for altering a targetSLAM sequence in a cell. The contiguous stretch of genomic DNA can bemodified by contacting the cell or population thereof with a Cas proteinor a nucleic acid encoding the Cas protein and at least one ribonucleicacid or at least one pair of ribonucleic acids selected from the groupconsisting of SEQ ID NOs: 350723-353590.

In some aspects, the present disclosure provides a stem cell orpopulation thereof comprising a genome in which the CD226 gene has beenedited to modify a contiguous stretch of genomic DNA. In certainaspects, the present disclosure provides a method for altering a targetCD226 sequence in a cell. The contiguous stretch of genomic DNA can bemodified by contacting the cell or population thereof with a Cas proteinor a nucleic acid encoding the Cas protein and at least one ribonucleicacid or at least one pair of ribonucleic acids selected from the groupconsisting of SEQ ID NOs: 353591-416840.

In some aspects, the present disclosure provides a stem cell orpopulation thereof comprising a genome in which the PD1 gene has beenedited to modify a contiguous stretch of genomic DNA. In certainaspects, the present disclosure provides a method for altering a targetPD1 sequence in a cell. The contiguous stretch of genomic DNA can bemodified by contacting the cell or population thereof with a Cas proteinor a nucleic acid encoding the Cas protein and at least one ribonucleicacid or at least one pair of ribonucleic acids selected from the groupconsisting of sequences numbered 196-531 and 4047-9101 in U.S.application Ser. No. 15/083,021, which is incorporated herein byreference.

In some aspects, the present disclosure provides a stem cell orpopulation thereof comprising a genome in which the CTLA4 gene has beenedited to modify a contiguous stretch of genomic DNA. In certainaspects, the present disclosure provides a method for altering a targetCTLA4 sequence in a cell. The contiguous stretch of genomic DNA can bemodified by contacting the cell or population thereof with a Cas proteinor a nucleic acid encoding the Cas protein and at least one ribonucleicacid or at least one pair of ribonucleic acids selected from the groupconsisting of sequences numbered 1-195 and 797-4046 in U.S. applicationSer. No. 15/083,021, which is incorporated herein by reference.

In some aspects, the present disclosure provides a stem cell orpopulation thereof comprising a genome in which the LAG3 gene has beenedited to modify a contiguous stretch of genomic DNA. In certainaspects, the present disclosure provides a method for altering a targetLAG3 sequence in a cell. The contiguous stretch of genomic DNA can bemodified by contacting the cell or population thereof with a Cas proteinor a nucleic acid encoding the Cas protein and at least one ribonucleicacid or at least one pair of ribonucleic acids selected from the groupconsisting of SEQ ID NOs: 416841-421195.

In some aspects, the present disclosure provides a stem cell orpopulation thereof comprising a genome in which the TIGIT gene has beenedited to modify a contiguous stretch of genomic DNA. In certainaspects, the present disclosure provides a method for altering a targetTIGIT sequence in a cell. The contiguous stretch of genomic DNA can bemodified by contacting the cell or population thereof with a Cas proteinor a nucleic acid encoding the Cas protein and at least one ribonucleicacid or at least one pair of ribonucleic acids selected from the groupconsisting of SEQ ID NOs: 421196-432039.

In some aspects, the present disclosure provides a stem cell orpopulation thereof comprising a genome in which the TIM3 gene has beenedited to modify a contiguous stretch of genomic DNA. In certainaspects, the present disclosure provides a method for altering a targetTIM3 sequence in a cell. The contiguous stretch of genomic DNA can bemodified by contacting the cell or population thereof with a Cas proteinor a nucleic acid encoding the Cas protein and at least one ribonucleicacid or at least one pair of ribonucleic acids selected from the groupconsisting of SEQ ID NOs: 432040-447610.

In some aspects, the present disclosure provides a stem cell orpopulation thereof comprising a genome in which the CD160 gene has beenedited to modify a contiguous stretch of genomic DNA. In certainaspects, the present disclosure provides a method for altering a targetCD160 sequence in a cell. The contiguous stretch of genomic DNA can bemodified by contacting the cell or population thereof with a Cas proteinor a nucleic acid encoding the Cas protein and at least one ribonucleicacid or at least one pair of ribonucleic acids selected from the groupconsisting of SEQ ID NOs: 447611-459294.

In some aspects, the present disclosure provides a stem cell orpopulation thereof comprising a genome in which the BTLA gene has beenedited to modify a contiguous stretch of genomic DNA. In certainaspects, the present disclosure provides a method for altering a targetBTLA sequence in a cell. The contiguous stretch of genomic DNA can bemodified by contacting the cell or population thereof with a Cas proteinor a nucleic acid encoding the Cas protein and at least one ribonucleicacid or at least one pair of ribonucleic acids selected from the groupconsisting of SEQ ID NOs: 459295-482454.

In some aspects, the present disclosure provides a stem cell orpopulation thereof comprising a genome in which the CD244 gene has beenedited to modify a contiguous stretch of genomic DNA. In certainaspects, the present disclosure provides a method for altering a targetCD244 sequence in a cell. The contiguous stretch of genomic DNA can bemodified by contacting the cell or population thereof with a Cas proteinor a nucleic acid encoding the Cas protein and at least one ribonucleicacid or at least one pair of ribonucleic acids selected from the groupconsisting of SEQ ID NOs: 482455-504169.

In some aspects, the present disclosure provides a stem cell orpopulation thereof comprising a genome in which the CD244 gene has beenedited to modify a contiguous stretch of genomic DNA. In certainaspects, the present disclosure provides a method for altering a targetCD244 sequence in a cell. The contiguous stretch of genomic DNA can bemodified by contacting the cell or population thereof with a Cas proteinor a nucleic acid encoding the Cas protein and at least one ribonucleicacid or at least one pair of ribonucleic acids selected from the groupconsisting of SEQ ID NOs: 482455-504169.

In some aspects, the present disclosure provides a stem cell orpopulation thereof comprising a genome in which the CD30 gene has beenedited to modify a contiguous stretch of genomic DNA. In certainaspects, the present disclosure provides a method for altering a targetCD30 sequence in a cell. The contiguous stretch of genomic DNA can bemodified by contacting the cell or population thereof with a Cas proteinor a nucleic acid encoding the Cas protein and at least one ribonucleicacid or at least one pair of ribonucleic acids selected from the groupconsisting of SEQ ID NOs: 699755-731993.

In some aspects, the present disclosure provides a stem cell orpopulation thereof comprising a genome in which the TLT gene has beenedited to modify a contiguous stretch of genomic DNA. In certainaspects, the present disclosure provides a method for altering a targetTLT sequence in a cell. The contiguous stretch of genomic DNA can bemodified by contacting the cell or population thereof with a Cas proteinor a nucleic acid encoding the Cas protein and at least one ribonucleicacid or at least one pair of ribonucleic acids selected from the groupconsisting of SEQ ID NOs: 731994-739957.

In some aspects, the present disclosure provides a stem cell orpopulation thereof comprising a genome in which the VISTA gene has beenedited to modify a contiguous stretch of genomic DNA. In certainaspects, the present disclosure provides a method for altering a targetVISTA sequence in a cell. The contiguous stretch of genomic DNA can bemodified by contacting the cell or population thereof with a Cas proteinor a nucleic acid encoding the Cas protein and at least one ribonucleicacid or at least one pair of ribonucleic acids selected from the groupconsisting of SEQ ID NOs: 739958-757515.

In some aspects, the present disclosure provides a stem cell orpopulation thereof comprising a genome in which the B7-H3 gene has beenedited to modify a contiguous stretch of genomic DNA. In certainaspects, the present disclosure provides a method for altering a targetB7-H3 sequence in a cell. The contiguous stretch of genomic DNA can bemodified by contacting the cell or population thereof with a Cas proteinor a nucleic acid encoding the Cas protein and at least one ribonucleicacid or at least one pair of ribonucleic acids selected from the groupconsisting of SEQ ID NOs: 757516-777888.

In some aspects, the present disclosure provides a stem cell orpopulation thereof comprising a genome in which the PD-L2 gene has beenedited to modify a contiguous stretch of genomic DNA. In certainaspects, the present disclosure provides a method for altering a targetPD-L2 sequence in a cell. The contiguous stretch of genomic DNA can bemodified by contacting the cell or population thereof with a Cas proteinor a nucleic acid encoding the Cas protein and at least one ribonucleicacid or at least one pair of ribonucleic acids selected from the groupconsisting of SEQ ID NOs: 777889-817976.

In some embodiments, lymphocyte adhesion is blocked to preventautoimmunity. In certain aspects, target genes can be edited by genomeediting to block lymphocyte adhesion (Table 5).

TABLE 5 Blocking lymphocyte adhesion to prevent autoimmunity. Ligand onCancer cells Receptor on T cells ICAM-1, ICAM-2 LFA-1 CD58/LFA-3 CD2CD-SIGN ICAM-3

In some aspects, the present disclosure provides a stem cell orpopulation thereof comprising a genome in which the LFA-1 gene has beenedited to modify a contiguous stretch of genomic DNA. In certainaspects, the present disclosure provides a method for altering a targetLFA-1 sequence in a cell. The contiguous stretch of genomic DNA can bemodified by contacting the cell or population thereof with a Cas proteinor a nucleic acid encoding the Cas protein and at least one ribonucleicacid or at least one pair of ribonucleic acids selected from the groupconsisting of SEQ ID NOs: 504170-526670.

In some aspects, the present disclosure provides a stem cell orpopulation thereof comprising a genome in which the CD2 gene has beenedited to modify a contiguous stretch of genomic DNA. In certainaspects, the present disclosure provides a method for altering a targetCD2 sequence in a cell. The contiguous stretch of genomic DNA can bemodified by contacting the cell or population thereof with a Cas proteinor a nucleic acid encoding the Cas protein and at least one ribonucleicacid or at least one pair of ribonucleic acids selected from the groupconsisting of SEQ ID NOs: 526671-536704.

In some aspects, the present disclosure provides a stem cell orpopulation thereof comprising a genome in which the CD58 gene has beenedited to modify a contiguous stretch of genomic DNA. In certainaspects, the present disclosure provides a method for altering a targetCD58 sequence in a cell. The contiguous stretch of genomic DNA can bemodified by contacting the cell or population thereof with a Cas proteinor a nucleic acid encoding the Cas protein and at least one ribonucleicacid or at least one pair of ribonucleic acids selected from the groupconsisting of SEQ ID NOs: 536705-570967.

In some embodiments, genes encoding for T cell receptors can be deletedby genome editing. Deletion of T cell receptor genes may occur toprevent autoimmune attack in T cell therapy. In some aspects, the geneencoding the T cell receptor is a T cell receptor alpha locus (TCRA), ora homolog, ortholog, or variant thereof (Gene ID: 5133, also known asIMD7, TCRD, TRA@, TRAC, and referred to herein as TCRa, TCRA, TCRalpha,and the like). In some aspects, the gene encoding the T cell receptor isa T cell receptor alpha locus (TCRB), or a homolog, ortholog, or variantthereof (Gene ID: 6957, also known as TCRB; TRB@, and referred to hereinas TCRb, TCRB, TCRbeta, and the like). In some aspects, the T cellreceptor gene is modified by genome editing as described in U.S.application Ser. No. 15/083,021 and PCT Application No.PCT/US2016/024554, both of which are incorporated herein by reference.

In some aspects, the present disclosure provides a stem cell orpopulation thereof comprising a genome in which the TRAC gene has beenedited to modify a contiguous stretch of genomic DNA. In certainaspects, the present disclosure provides a method for altering a targetTRAC sequence in a cell. The contiguous stretch of genomic DNA can bemodified by contacting the cell or population thereof with a Cas proteinor a nucleic acid encoding the Cas protein and at least one ribonucleicacid or at least one pair of ribonucleic acids selected from the groupconsisting of sequences numbered 532-609 and 9102-9797, as described inU.S. application Ser. No. 15/083,021, incorporated herein by reference.

In some aspects, the present disclosure provides a stem cell orpopulation thereof comprising a genome in which the TRBC gene has beenedited to modify a contiguous stretch of genomic DNA. In certainaspects, the present disclosure provides a method for altering a targetTRBC sequence in a cell. The contiguous stretch of genomic DNA can bemodified by contacting the cell or population thereof with a Cas proteinor a nucleic acid encoding the Cas protein and at least one ribonucleicacid or at least one pair of ribonucleic acids selected from the groupconsisting of sequences numbered 610-765 and 9798-10573, as described inU.S. application Ser. No. 15/083,021 and incorporated herein byreference.

In some embodiments, genes involved in regulatory T cell (T reg)function can be deleted by genome editing (Table 6). Deletion of genesinvolved in regulatory T cell (T reg) function may occur to breaktolerance in T cell therapy.

TABLE 6 Deletion of genes involved in regulatory T cell (T reg) functionto break tolerance in T cell therapy. Gene Function FOXP3 T regdevelopment HELIOS T reg maintenance ST2 IL-3 3 receptor

In some aspects, the present disclosure provides a stem cell orpopulation thereof comprising a genome in which the FOXP3 gene has beenedited to modify (e.g., delete) a contiguous stretch of genomic DNA. Incertain aspects, the present disclosure provides a method for altering atarget FOXP3 sequence in a cell. The contiguous stretch of genomic DNAcan be modified (e.g., deleted) by contacting the cell or populationthereof with a Cas protein or a nucleic acid encoding the Cas proteinand at least one ribonucleic acid or at least one pair of ribonucleicacids selected from the group consisting of SEQ ID NOs: 570968-584068.

In some aspects, the present disclosure provides a stem cell orpopulation thereof comprising a genome in which the HELIOS gene has beenedited to modify a contiguous stretch of genomic DNA. In certainaspects, the present disclosure provides a method for altering a targetHELIOS sequence in a cell. The contiguous stretch of genomic DNA can bemodified by contacting the cell or population thereof with a Cas proteinor a nucleic acid encoding the Cas protein and at least one ribonucleicacid or at least one pair of ribonucleic acids selected from the groupconsisting of SEQ ID NOs: 683033-688807.

The capacity of the CRISPR/Cas system for multiplexing also allows thegeneration of disease-specific universal donor cell lines that harborone or more genomic alterations that will improve their applicability totreat a certain disease or condition. A list of potential diseases thatcan be addressed can be found in Table 7.

TABLE 7 Additional targets that can be modified using the CRISPR/Cassystem in order to generate universal donor cell lines tailored to aspecific disease or application. Gene Disease TCR T cell therapy PD-1 Tcell therapy CTLA4 T cell therapy LAG-3 T cell therapy TIGIT T celltherapy TIM3 T cell therapy CCR5 HIV resistance PCSK9 Cardiovasculardisease APOC3 Cardiovascular disease

It is to be understood that the inventions disclosed herein are notlimited in their application to the details set forth in the descriptionor as exemplified. The invention encompasses other embodiments and iscapable of being practiced or carried out in various ways. Also, it isto be understood that the phraseology and terminology employed herein isfor the purpose of description and should not be regarded as limiting.

While certain compositions, methods and assays of the present inventionhave been described with specificity in accordance with certainembodiments, the following examples serve only to illustrate the methodsand compositions of the invention and are not intended to limit thesame.

The articles “a” and “an” as used herein in the specification and in theclaims, unless clearly indicated to the contrary, should be understoodto include the plural referents. Claims or descriptions that include“or” between one or more members of a group are considered satisfied ifone, more than one, or all of the group members are present in, employedin, or otherwise relevant to a given product or process unless indicatedto the contrary or otherwise evident from the context. The inventionincludes embodiments in which exactly one member of the group is presentin, employed in, or otherwise relevant to a given product or process.The invention also includes embodiments in which more than one, or theentire group members are present in, employed in, or otherwise relevantto a given product or process. Furthermore, it is to be understood thatthe invention encompasses all variations, combinations, and permutationsin which one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the listed claims is introduced into anotherclaim dependent on the same base claim (or, as relevant, any otherclaim) unless otherwise indicated or unless it would be evident to oneof ordinary skill in the art that a contradiction or inconsistency wouldarise. Where elements are presented as lists, (e.g., in Markush group orsimilar format) it is to be understood that each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should be understood that, in general, where the invention, oraspects of the invention, is/are referred to as comprising particularelements, features, etc., certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements, features, etc. For purposes of simplicity those embodimentshave not in every case been specifically set forth in so many wordsherein. It should also be understood that any embodiment or aspect ofthe invention can be explicitly excluded from the claims, regardless ofwhether the specific exclusion is recited in the specification. Thepublications and other reference materials referenced herein to describethe background of the invention and to provide additional detailregarding its practice are hereby incorporated by reference.

Examples

Cell Lines

The inventors have targeted various genes in a variety of cell lines.The various cell lines utilized include HuES8, HuES9, BJ-RiPSCs, Thp1,Jurkat, Primary T cells and HEK293T cells (FIG. 27 ). HuES8 and HuES9are human ES cell lines. BJ-RiPSC is an iPSC line.

Knock-Out of HLA-A, HLA-B, and HLA-C

Knock-out targeting of HLAs was examined Initially, an HLA-B and HLA-Cknock-out strategy was examined Two short guide RNAs (sgRNAs) weredesigned upstream of the HLA-B locus and downstream of HLA-C, whichallowed for excision of the HLA-B and HLA-C genes (FIG. 14A). sgRNA #1and sgRNA #2 target the HLA-B upstream region, and sgRNA #3 and sgRNA #4target the HLA-C downstream region.

PCR screening was performed and confirmed that clone 1D was a homozygousknock-out clone. A schematic showing the PCR verification strategy forsuccessful deletion of HLA-B and HLA-C is shown in FIG. 13B. Two pairsof wild-type (WT) primers were designed flanking each cutting site, withpredicted amplicons sizes of 545 bp and 472 bp. Clone 1D was identifiedas a homozygous knock-out clone by the presence of ˜680 bp PCR bandgenerated with KO primers and the absence of bands using the twodifferent sets of WT primers (FIG. 14B). In addition, genomic DNA wasisolated from the indicated targeted HuES8 clones. Sequencing results ofthe clone 1D PCR product demonstrated successful deletion of HLA-B andHLA-C genes in HuES9 (FIG. 14C).

Further, RT-PCR with HLA-B and HLA-C specific primers demonstrated themRNA expression of HLA-B and HLA-C in clone 1D was eliminated (FIG.14D). GAPDH, TOP1 and HPRT1 were used as internal controls. WT and clone1D RT-PCR products amplified with HLA-B primers were sequenced andidentified as HLA-B and HLA-A mRNAs, respectively using BLAST (FIG.14E). These results demonstrated that in the absence of the HLA-B gene,the HLA-B specific primers will amplify HLA-A mRNA in HuES8 clone 1D.HuES8 clone 1D displays a normal karyotype as assessed by NanoStringnCounter set (FIG. 14F).

A schematic demonstrating an HLA-A knockout strategy using the dualsgRNA approach is provided in FIG. 14G. The schematic shows the positionof the two sgRNAs (#5 and #6) that were designed to bind upstream anddownstream of HLA-A. The on-target cutting efficiency of sgRNA #5 and #6was determined in 293T cells using TIDE (FIG. 14H).

PCR screening confirmed that clone 4E was a heterozygous HLA-A knockoutclone (FIG. 14I). The PCR screening strategy confirmed deletion of HLA-Ain HuES8. KO primers were designed with one primer annealing upstreamand one primer annealing downstream of the cutting sites. Upon HLA-Adeletion, the resulting amplicon was observed as 220 bp on a 2% agarosegel. Two pairs of WT primers were designed flanking each cutting site,with predicted amplicon sizes of 589 bp and 571 bp. Clone 4E wasidentified as a heterozygous clone due to the presence of bandsgenerated with KO primers and WT primers amplified from genomic DNA(FIG. 14I). Sequencing of the PCR product amplified from the genomic DNAof clone 4E using KO primers demonstrated successful deletion of HLA-Ain HuES8 (FIG. 14J).

Knock-Out of Transcriptional Regulators

As illustrated in FIGS. 3A-3B, in certain aspects, the inventionsdisclosed herein target the transcriptional regulators of antigenpresentation (e.g., CIITA and/or NLRC5). The inventors have discoveredthat targeting and/or modulating the expression of such transcriptionalregulators of antigen presentation provides a means to efficientlymodulate (e.g., reduce or eliminate) the expression of the threeclassical MHC-Ia molecules, HLA-A, HLA-B, and HLA-C, and thereby producehypoimmunogenic universal stem cell lines that are useful for cellreplacement therapy. As evidenced in FIG. 4 , the present inventors havedemonstrated that such genome-edited cells are characterized by reducedMHC-I expression relative to the WT HuES9 cells, as assessed by FACS.Reduced or eliminated MHC-I surface expression was observed in thosestem cells in which the expression of NLRC5, CIITA and B2M was modulatedusing the CRISPR/Cas system. In particular, FIG. 4 evidences low basalMHC-I expression in stem cells which can be increased by IFN-γ, that anapproximately 50% reduction of MHC-I surface expression was observed inthe IFN-γ-treated NLRC5^(−/−) stem cells and that a complete loss ofMHC-I surface expression was observed in the B2M^(−/−) stem cells.

Similar results were observed on MHC-II expression following modulationof the expression of CIITA. The present inventors differentiated thegenome-edited CIITA stem cell line into macrophage, which are antigenpresenting cells. As depicted in FIG. 5C, the CIITA-deficient stemcell-derived macrophage lacked MHC-II expression, thus clearlyshowcasing the expected phenotype. In particular, by targeting the firstcoding exon of the CIITA gene (FIGS. 10A-10B), the foregoing resultsevidence that MHC-II expression can be efficiently abrogated.

The functional consequences of reduced HLA expression in thegenome-edited cells were next evaluated in a humanized mouse model thatwas prepared by reconstituting an immunocompromised mice model with ahuman immune system (FIG. 6 ). As illustrated in Table 1 below, acomparable number of teratomas formed in all cases, irrespective of thegenotype and the whole colony had to be sacrificed. Similarly, nodifferences in the failure rates of teratoma formation was observed. Theinventors looked more closely at the overall morphology of the teratomasamples and a clear cut phenotype that correlates with the levels ofMHC-I expression was identified. NLRC5, reduced, B2M absent MHC-I on thesurface. FIG. 7 illustrates the quantification of the resulting teratomatypes. FIGS. 8A-8C further demonstrate CD8+ T cell proliferation inwild-type cells and suggests immune rejection of the wild-type teratomasand improved engraftment of the genome-edited cells in the humanizedmouse model.

TABLE 1 Genotype #tumors (injections) p42 WT 21 (24) E4 NLRC5−/−CIITA−/−21 (24) C7 B2M−/−CIITA−/− 20 (24) C6 NLRC5−/−CIITA−/− 18 (20)

The foregoing results therefore demonstrate successful targeting ofNLRC5, CIITA and B2M in HuES9 and BJ RiPSCs and further evidence thatgenome-edited cells can be differentiated into a variety of differentcell types that are characterized by reduced HLA expression.

The inventors have used both a TALENs system and a CRISPR/Cas system tofacilitate the targeting of transcriptional regulators of HLA expression(i.e., the MHC enhanceosome). TALEN-induced CIITA and NLRC5 mutations inBJ-RIPSCs and HuES9 are illustrated in FIGS. 15A-D. In addition, NLRC5and CIITA can also be targeted utilizing CRISPRs to achieve a reductionin MHC class I expression and complete loss of MHC class II expression,respectively.

NLRC5 was targeted in Thp1 cells using CRISPR and it was demonstratedthat MHC-I expression in NLRC5−/− Thp1 cells was reduced (FIG. 16A).Examples of CRISPR and/or TALENs systems targeting NLRC5, CIITA and B2Mare provided in FIG. 16B. Reduced MHC class I expression in HuES9 wasshown following targeting with NLRC5 or B2M CRISPRs. For example, about50% reduction was shown in IFNγ-treated NLRC5−/− cells and complete lossof MHC-I surface expression was shown in B2M−/− cells (FIG. 16C).

A lentiviral transduction of Thp-1 was conducted using a two component(e.g., dual vector) system (FIG. 16D). The two component system includedLenti-Cas9-Blasticidin and Lenti-Guide-puro. Examples of the differentCRISPRs used for the lentiviral transduction of Thp-1 are provided inFIG. 16E. Thp-1 was transduced with lentivirus encoding NLRC5 and CIITA.A B2M CRISPR was used as a positive control. All of the cells werestimulated ON with 50U IFNγ to boost HLA expression (HLA-A2 was 1:200and HLA-DR was 1:100). It was demonstrated that CIITA and NLRC5 actindependently on MHC-II and MHC-I, respectively (FIG. 16F). At ten dayspost CRISPR transduction, when the Thp1 cells were all stimulated ONwith 50U IFNγ (HLA-A2 is 1:200 and HLA-DR is 1:100), it was demonstratedthat targeting IRF1 results in a loss of MHC-II expression (FIG. 16G).

The inventors further examined targeting of IRF1 and the resultantreduced MHC class I expression in human pluripotent stem cells (HuES9)and Thp-1 cells. A schematic demonstrating a dual CRISPR strategy fortargeting the IRF1 locus is provided in FIG. 17A. Testing of differentIRF1 guide combinations was conducted (FIG. 17B) and a ‘dual guidestrategy’ for the targeted deletion of IRF1 was identified (FIG. 17C).After the dual guide strategy for the targeted deletion of IRF1 wasapplied, a sequence confirmation of IRF1 CRISPR induced deletion wasprovided (FIG. 17D), followed by screening of IRF-1 targeted HuES9 cells(FIG. 17E). The presence of the PCR bands suggests successful targetingusing the dual CRISPR strategy.

The inventors then reconfirmed (FIG. 17F) and genotyped (FIG. 17G) theIRF1 clones. Sequencing confirmed IRF1 CRISPR induced deletion in clone12 (FIG. 17H), clone 17 (FIG. 17I) and clone 21 (FIG. 17J). The IRF1−/−HuES9 clones exhibited impaired MHC class I induction following IFNγtreatment for 48 hours (FIG. 17K).

The inventors also examined targeting of additional enhanceosomecomponents. The impact on MHC-I expression by targeting additionalenhanceosome components in 293T cells was examined, but it is expectedthat the targeting of these additional enhanceosome components will alsoaffect MHC-II levels in cells that actually express it, e.g., Thp1 cellsor other APCs. For example, the inventors examined CRISPR targeting ofRFX5 (FIG. 18A), RFX-ANK (FIG. 18C) and RFK-AP (FIG. 18G) in 293T cellsusing a dual guide strategy. The targeting results demonstrated reducedMHC class I expression of RFX5 (FIG. 18B, FIGS. 18E-18F), RFK-ANK (FIG.18D, FIGS. 18E-F) and RFK-AP (FIG. 18H). The inventors also examinedCRISPR dual guide targeting of NFY-A (FIG. 19A), NFY-B (FIG. 19D) andNFY-C (FIG. 19B) in 293T cells. The targeting results demonstratedreduced MHC class I expression of NFY-A (FIG. 19C), NFY-B (FIG. 19E) andNFY-C (FIG. 19C).

Blocking Surface Trafficking of MHC Class I

The successful targeting of B2M utilizing TALENs and/or CRISPR haspreviously been described in PCT Patent Application No.PCT/US2015/059621, which is incorporated herein by reference and isshown in FIGS. 32-40 . It has further been demonstrated that surfaceexpression of MHC class I molecules is reduced or eliminated, therebyblocking the surface trafficking of MHC-I molecules. For example, lossof MHC class I surface expression in B2M−/− Jurkat Cas9 T cells wasshown in comparison to MHC class I surface expression in Jurkat Cas9 Tcells in FIG. 31 .

In addition, the inventors have demonstrated that surface trafficking ofMHC class I molecules can be suppressed but disrupting the TAP1 gene, anER-resident peptide transporter. For example, TAP1 CRISPR expressionreduces MHC-I surface expression in Jurkat cells (FIG. 20A), as well asin Jurkat T cells (FIG. 20B), following 48 hour treatment with IFNγ. Itwas further demonstrated that HLA surface expression in Jurkat (Cas9) Tcells could be eliminated (FIG. 20C and FIG. 20D). The Jurkat T cellswere established from the peripheral blood of a 14 year old boy who hadacute T cell leukemia.

HLA Razor

The inventors examined CRISPR guide RNAs that allow simultaneousdeletion of all MHC class I alleles by targeting a conserved region inthe HLA genes. A TALEN pair of guides that target the same conservedregion in MHC class I genes were also identified. Any guide that targetsthe conserved regions may be identified or classified as an HLA Razor.The targeting of a conserved sequence found in all HLAs by CRISPR orTALENS was demonstrated in FIG. 21A. The two violet boxes indicate thebinding sites for the pan-HLA TALEN pair. The blue arrow indicates thepan-HLA CRISPR tested pair, with the PAM being boxed in blue. Theexpression of pan-HLA-TALENs in 293T and HuES9 cells was shown in FIG.21B at 72 hours post transfection. The HLA-Razor CRISPR blunts MHC classI expression in 293T cells as demonstrated in FIG. 21C. The 293T cellswere also co-transfected with Cas9-GFP. FIG. 21D provides a comparisonof the activity of two different HLA Razors targeting two differentconserved regions of the HLAs.

Knock-In of PD-L1 and HLA-G

The present inventors have used a CRISPR/Cas system to facilitate theinsertion of tolerogenic factors, such as the tolerogenic factors shownin Table 2, into an AAVS1 locus, to actively inhibit immune rejection.As evidenced in FIGS. 11A-11C, tolerogenic factors, such as PD-L1 andHLA-G, have been successfully expressed from the AAVS1 locus.

A schematic of the PD-L1 and HLA-G knock-in strategy is shown in FIG.22A. Wild type (WT) and knock-in (KI) primers for clone screening weredesigned. The amplicon with WT primers is predicted as 488 bp, and theamplicons with KI primers are predicted as 403 bp and 915 bp. A designof a knock-in donor plasmid (FIG. 22B) shows that the reading frames ofPD-L1 and HLA-G are linked by T2A and their expression is driven by aCAGGS promoter. Puromycin was used as a drug resistance marker followingthe SA-2A gene trap element.

The expression of PD-L1 and HLA-G was examined in the donorplasmid-transfected 293T cells by FACS analysis. FIG. 22C shows theectopic PD-L1 and HLA-G expression in 293T cells. APC-conjugated PD-L1antibody and FITC-conjugated HLA-G antibody were used. In addition, theexpression of ectopic HLA-G expression in JEG-3 cells is shown in FIG.22D. The donor plasmid was transfected into an HLA-G−/− JEG-3 cell line,and ectopic HLA-G expression was examined by FACS analysis 48 hourspost-transfection. A PE-conjugated HLA-G antibody (MEM/G9) was used todetect surface HLA-G surface expression.

PCR screening was performed, which confirmed that clone 1G was aheterozygous KI clone for PD-L1/HLA-G (FIG. 22E). Clone 1G wasidentified as a heterozygous KI clone by the presence of bands usingboth WT primers and KI-specific primers amplified from genomic DNA oftargeted HuES8. The expression of PD-L1 was verified in HuES8 KI clone1G by FACS analysis using an APC-conjugated PD-L1 antibody (FIG. 22F).

Knock-In of CD-47

In addition, a CD47 knock-in strategy was also examined Human CD47 wascloned into an expression plasmid driven by a CAG promoter, which alsocontained an IRES-GFP. 293T cells already express high levels of CD47(the human “don't eat me” signal), which prevents engulfment of cellsfrom macrophages. Expression can be increased by overexpression of CD47in both 293T cells, as well as in human pluripotent stem cells (HuES9),as detected by FACS using a CD47-specific antibody (FIG. 23 ). It isexpected that overexpression of CD47 will assist engraftment of stemcell-derived transplants by protecting cells from macrophage engulfment.

Additional Targets to be Modified in Universal Donor Cells

Additional targets may be modified in universal donor cells to tailorthem to a specific application. For example, additional targets may bemodified in universal donor cells to tailor them to be used as universalCAR T cells. In one instance, the inventors deleted TRAC and TRBC inHuES9 to disrupt TCR expression (FIG. 24 ). A dual guide RNA approachwas used to introduce deletions into the TRAC and TRBC loci in HuES9cells. The TCRA wild type band is 249 bp and after deletion is 209 bp.The TCRB wild type band is 162 bp and after deletion is 140 bp. Theinventors additionally targeted TCRA in HuES9 B2M−/−CIITA−/− to create atriple knock-out stem cell line for B2M−/−, CIITA−/− and TCR−/−, whichupon differentiation into T cells will be devoid of MHC-I and -II andexhibit no TCR surface expression.

In another instance, the inventors targeted PD-L1 (FIG. 25A). It wasidentified that CRISPRs targeting Cd274/B7-H1/PD-L1 were very useful inbreaking tolerance in cancer immunotherapy. PD-L1 knock out wasdemonstrated in multiple cell types, including JEG-3, a choriocarcinomacell line, and in two melanoma cell lines, 501 and MalMe (FIGS.25B-25D). Screening of targeted B7-H1 colonies was conducted, and it wasidentified that there was an 8.3% CRISPR cutting efficiency in JEG-3cells (FIG. 25B). Additionally, reconfirmation of 501 melanoma knock outclones was shown in FIG. 25C, and reconfirmation of MalMe melanoma knockout clones was shown in FIG. 25D.

Targeting Co-Inhibitor/Co-Stimulatory Receptors or their Ligands

The inventors examined multiple targets utilizing CRISPR. Anillustration of co-stimulatory/inhibitory molecules and their receptorson T cells is provided in FIG. 26A. For each target examined, theindicated four CRISPRs have been cloned and tested for on-targetactivity in 293T cells. The expected size of the PCR band, when cuttingof both CRISPRs occurs, is indicated below each individual gel picture(FIGS. 26B-26P). Dual guide targeting in 293T cells was demonstrated forTIGIT (FIG. 26B), TIM3 (FIG. 26C), HVEM (FIG. 26D), 2B4/CD244 (FIG.26E), CD28 (FIG. 26F), OX40 (FIG. 26G), B71 (FIG. 26H), CD226 (FIG.26I), CD2 (FIG. 26J), LAG3 (FIG. 26K), BTLA (FIG. 26L), ICOS (FIG. 26M),CD27 (FIG. 26N), ST2 (FIG. 26O) and GITR (FIG. 26P).

CRISPR targeting of B7-H3 was examined in JEG-3 cells (FIGS. 30A-30D).FIG. 30B shows a screening of targeted B7-H3 colonies in JEG-3 cells andidentifies a CRISPR cutting efficiency of 15/80 or about 18.7%.Confirmation of the B7-H3 knock-outs through sequencing was performed(FIG. 30C) and the loss of B7-H3 surface expression in targeted Jeg3clones was provided in FIG. 30D.

Differentiation of Modified Embryonic Stem Cells

Modified embryonic stem cells may be differentiated into a variety ofdifferent cell types, with reduced or absent HLA expression (FIG. 28A).Examples of such cell types include mesenchymal progenitors cells(MPCs), hypoimmunogenic cardiomyocytes, endothelial cells (ECs),macrophages, hepatocytes, beta cells (e.g., pancreatic beta cells), orneural progenitor cells (NPCs).

Initially, the inventors demonstrated reduced MHC-I expression inNLRC5−/− human ES cells in FIG. 28B. Low basal MHC-I expression was seenin stem cells, but expression could be increased by IFNγ stimulation.There is about a 50% reduction in IFNγ-treated NLRC5−/− cells.

The inventors then examined MHC-I expression in various differentiatedcell types. For example, FIG. 28C shows reduced MHC-I expression inNLRC5−/− human mesenchymal progenitor cells (MPCs). The graphs includedin FIG. 28C demonstrate the differences between MHC-I expression inHuES9 cells and MPC cells. FIG. 28D shows reduced MHC-I expression instem cell-derived NLRC5−/− endothelial cells (ECs). Similardifferentiation efficiency for ECs was shown in FIG. 28E, and a loss ofHLA expression in B2M−/−CIITA−/− ECs was shown in FIG. 28F. Next,reduced MHC class I expression in NLRC5−/− hepatocyte-like cells (HLCs)was provided (FIG. 28G). The HLCs were derived from BJ-RIPSCs.

At FIG. 28H it is shown that a mutation of CIITA abrogates MHC class IIexpression in hESC-derived macrophages. Additionally, neural progenitorcells (NPCs) are differentiated from an embryonic stem cell (FIG. 28I).It was shown that B2M−/−CIITA−/−HuES9 cells form Nestin+ neural rosettes(white arrows in figure) as a result of differentiation. Finally, theinventors have adapted modified embryonic stem cells to spin culture soas to be utilized for beta-cell differentiation (FIG. 28J).

In Vivo Data

Finally, the inventors have generated ‘hypoimmunogenic’ DKO cell lines(FIG. 29A). The cells lines examined were WT HuES9 cells, NLRC5−/−C2TA−/− HuES9 cells and B2M−/− C2TA−/− HuES9 cells. The different cellslines showed different levels of MHC-I and MHC-II expression (FIG. 29A).For example, WT HuES9 cells exhibited expression of MHC-I and MHC-II.The NLRC5−/− C2TA−/− HuES9 cells exhibited reduced MHC-I expression andno MHC-II expression. The B2M−/− C2TA−/− HuES9 cells exhibited no MHC-Iand MHC-II expression. These cells lines were then examined in humanizedmice and improved engraftment of genome-edited stem cells in humanizedmice was shown in FIG. 29B.

What is claimed is:
 1. A genetically modified stem cell comprisingmodulated expression of one or more MHC-I human leukocyte antigenmolecules and one or more MHC-II human leukocyte antigen moleculesrelative to a wild-type stem cell; and increased expression of one ormore tolerogenic factors relative to a wild-type stem cell, wherein anucleic acid encoding the one or more tolerogenic factors is insertedinto a safe harbor locus of at least one allele of the cell, wherein thestem cell is the same type of cell as the wild-type stem cell.
 2. Thestem cell of claim 1, wherein the safe harbor locus comprises an AAVS1locus.
 3. The stem cell of claim 1, wherein the one or more tolerogenicfactors inhibit immune rejection.
 4. The stem cell of claim 1, whereinthe one or more tolerogenic factors are selected from the groupconsisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, CD47, C1-inhibitor,and IL-35.
 5. The stem cell of claim 1, wherein the one or moretolerogenic factors consist of CD47.
 6. The stem cell of claim 1,wherein the one or more tolerogenic factors consist of HLA-C.
 7. Thestem cell of claim 1, wherein the one or more tolerogenic factorsconsist of HLA-E.
 8. The stem cell of claim 1, wherein the one or moretolerogenic factors consist of HLA-G.
 9. The stem cell of claim 1,wherein the one or more tolerogenic factors consist of PD-L1.
 10. Thestem cell of claim 1, wherein the one or more tolerogenic factorsconsist of CTLA-4-Ig.
 11. The stem cell of claim 1, wherein the one ormore tolerogenic factors consist of C1-inhibitor.
 12. The stem cell ofclaim 1, wherein the one or more tolerogenic factors consist of IL-35.13. The stem cell of claim 1, further comprising a modified genomecomprising a genomic modification wherein the β-2 microglobulin (β2M)gene is edited to reduce or eliminate MHC-I molecule surface expression.14. The stem cell of claim 13, wherein the genomic modificationcomprises knocking out expression of β2M and/or TAP1.
 15. The stem cellof claim 1, wherein the modulated expression of the MHC-I humanleukocyte antigen molecules or the MHC-II human leukocyte antigenmolecules results from one or more indels being introduced into one ormore genes encoding one or more human leukocyte antigens.
 16. The stemcell of claim 15, wherein the modulated expression of the MHC-I humanleukocyte antigen molecules results from one or more indels beingintroduced into HLA-A, HLA-B, HLA-C or a combination thereof.
 17. Thestem cell of claim 16, wherein the modulated expression of the MHC-IIhuman leukocyte antigen molecules results from one or more indels beingintroduced into class II major histocompatibility complex transactivator(CIITA).
 18. The stem cell of claim 1, wherein the modulated expressionof the MHC-I human leukocyte antigen molecules or the MHC-II humanleukocyte antigen molecules results from one or more indels beingintroduced into one or more genes encoding one or more transcriptionalregulators of MHC-I or MHC-II.
 19. The stem cell of claim 18, whereinthe transcriptional regulators are selected from the group consisting ofCIITA, β2M, TAP I, NLRC5, RFX5, RFXAP, RFXANK, NFY-A, NFY-B, NFY-C,IRF-1 and combinations thereof.
 20. The stem cell of claim 19, whereinthe stem cell comprises a CIITA knock out.
 21. The stem cell of claim 1,wherein the stem cell is a CIITA^(−/−) stem cell.
 22. The stem cell ofclaim 19, wherein the stem cell comprises a β2M knock out.
 23. The stemcell of claim 1, wherein the stem cell is a β2M^(−/−) stem cell.
 24. Thestem cell of claim 23, wherein the stem cell is a β2M^(−/−) CIITA^(−/−)stem cell.
 25. The stem cell of claim 1, wherein the stem cell is anembryonic stem cell.
 26. The stem cell of claim 1, wherein the stem cellis an induced pluripotent stem cell.
 27. The stem cell of claim 15,wherein the one or more tolerogenic factors are selected from the groupconsisting of HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, CD47, C1-inhibitor,and IL-35.
 28. The stem cell of claim 25, wherein the one or moretolerogenic factors are selected from the group consisting of HLA-C,HLA-E, HLA-G, PD-L1, CTLA-4-Ig, CD47, C1-inhibitor, and IL-35.
 29. Thestem cell of claim 26, wherein the one or more tolerogenic factors areselected from the group consisting of HLA-C, HLA-E, HLA-G, PD-L1,CTLA-4-Ig, CD47, C1-inhibitor, and IL-35.